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1.  Ranking the selectivity of PubChem screening hits by activity-based protein profiling: MMP13 as a case study 
Bioorganic & medicinal chemistry  2008;17(3):1101-1108.
High-throughput screening (HTS) has become an integral part of academic and industrial efforts aimed at developing new chemical probes and drugs. These screens typically generate several “hits”, or lead active compounds, that must be prioritized for follow-up medicinal chemistry studies. Among primary considerations for ranking lead compounds is selectivity for the intended target, especially among mechanistically related proteins. Here, we show how the chemical proteomic technology activity-based protein profiling (ABPP) can serve as a universal assay to rank HTS hits based on their selectivity across many members of an enzyme superfamily. As a case study, four metalloproteinase-13 (MMP13) inhibitors of similar potency originating from a publically supported HTS and reported in PubChem were tested by ABPP for selectivity against a panel of 27 diverse metalloproteases. The inhibitors could be readily separated into two groups: 1) those that were active against several metalloproteases, and 2) those that showed high selectivity for MMP13. The latter set of inhibitors was thereby designated as more suitable for future medicinal chemistry optimization. We anticipate that ABPP will find general utility as a platform to rank the selectivity of lead compounds emerging from HTS assays for a wide variety of enzymes.
PMCID: PMC2661618  PMID: 18364257
2.  Analysis of multiple compound–protein interactions reveals novel bioactive molecules 
The authors use machine learning of compound-protein interactions to explore drug polypharmacology and to efficiently identify bioactive ligands, including novel scaffold-hopping compounds for two pharmaceutically important protein families: G-protein coupled receptors and protein kinases.
We have demonstrated that machine learning of multiple compound–protein interactions is useful for efficient ligand screening and for assessing drug polypharmacology.This approach successfully identified novel scaffold-hopping compounds for two pharmaceutically important protein families: G-protein-coupled receptors and protein kinases.These bioactive compounds were not detected by existing computational ligand-screening methods in comparative studies.The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.
The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. Perturbations of biological systems by chemical probes provide broader applications not only for analysis of complex systems but also for intentional manipulations of these systems. Nevertheless, the lack of well-characterized chemical modulators has limited their use. Recently, chemical genomics has emerged as a promising area of research applicable to the exploration of novel bioactive molecules, and researchers are currently striving toward the identification of all possible ligands for all target protein families (Wang et al, 2009). Chemical genomics studies have shown that patterns of compound–protein interactions (CPIs) are too diverse to be understood as simple one-to-one events. There is an urgent need to develop appropriate data mining methods for characterizing and visualizing the full complexity of interactions between chemical space and biological systems. However, no existing screening approach has so far succeeded in identifying novel bioactive compounds using multiple interactions among compounds and target proteins.
High-throughput screening (HTS) and computational screening have greatly aided in the identification of early lead compounds for drug discovery. However, the large number of assays required for HTS to identify drugs that target multiple proteins render this process very costly and time-consuming. Therefore, interest in using in silico strategies for screening has increased. The most common computational approaches, ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS; Oprea and Matter, 2004; Muegge and Oloff, 2006; McInnes, 2007; Figure 1A), have been used for practical drug development. LBVS aims to identify molecules that are very similar to known active molecules and generally has difficulty identifying compounds with novel structural scaffolds that differ from reference molecules. The other popular strategy, SBVS, is constrained by the number of three-dimensional crystallographic structures available. To circumvent these limitations, we have shown that a new computational screening strategy, chemical genomics-based virtual screening (CGBVS), has the potential to identify novel, scaffold-hopping compounds and assess their polypharmacology by using a machine-learning method to recognize conserved molecular patterns in comprehensive CPI data sets.
The CGBVS strategy used in this study was made up of five steps: CPI data collection, descriptor calculation, representation of interaction vectors, predictive model construction using training data sets, and predictions from test data (Figure 1A). Importantly, step 1, the construction of a data set of chemical structures and protein sequences for known CPIs, did not require the three-dimensional protein structures needed for SBVS. In step 2, compound structures and protein sequences were converted into numerical descriptors. These descriptors were used to construct chemical or biological spaces in which decreasing distance between vectors corresponded to increasing similarity of compound structures or protein sequences. In step 3, we represented multiple CPI patterns by concatenating these chemical and protein descriptors. Using these interaction vectors, we could quantify the similarity of molecular interactions for compound–protein pairs, despite the fact that the ligand and protein similarity maps differed substantially. In step 4, concatenated vectors for CPI pairs (positive samples) and non-interacting pairs (negative samples) were input into an established machine-learning method. In the final step, the classifier constructed using training sets was applied to test data.
To evaluate the predictive value of CGBVS, we first compared its performance with that of LBVS by fivefold cross-validation. CGBVS performed with considerably higher accuracy (91.9%) than did LBVS (84.4%; Figure 1B). We next compared CGBVS and SBVS in a retrospective virtual screening based on the human β2-adrenergic receptor (ADRB2). Figure 1C shows that CGBVS provided higher hit rates than did SBVS. These results suggest that CGBVS is more successful than conventional approaches for prediction of CPIs.
We then evaluated the ability of the CGBVS method to predict the polypharmacology of ADRB2 by attempting to identify novel ADRB2 ligands from a group of G-protein-coupled receptor (GPCR) ligands. We ranked the prediction scores for the interactions of 826 reported GPCR ligands with ADRB2 and then analyzed the 50 highest-ranked compounds in greater detail. Of 21 commercially available compounds, 11 showed ADRB2-binding activity and were not previously reported to be ADRB2 ligands. These compounds included ligands not only for aminergic receptors but also for neuropeptide Y-type 1 receptors (NPY1R), which have low protein homology to ADRB2. Most ligands we identified were not detected by LBVS and SBVS, which suggests that only CGBVS could identify this unexpected cross-reaction for a ligand developed as a target to a peptidergic receptor.
The true value of CGBVS in drug discovery must be tested by assessing whether this method can identify scaffold-hopping lead compounds from a set of compounds that is structurally more diverse. To assess this ability, we analyzed 11 500 commercially available compounds to predict compounds likely to bind to two GPCRs and two protein kinases. Functional assays revealed that nine ADRB2 ligands, three NPY1R ligands, five epidermal growth factor receptor (EGFR) inhibitors, and two cyclin-dependent kinase 2 (CDK2) inhibitors were concentrated in the top-ranked compounds (hit rate=30, 15, 25, and 10%, respectively). We also evaluated the extent of scaffold hopping achieved in the identification of these novel ligands. One ADRB2 ligand, two NPY1R ligands, and one CDK2 inhibitor exhibited scaffold hopping (Figure 4), indicating that CGBVS can use this characteristic to rationally predict novel lead compounds, a crucial and very difficult step in drug discovery. This feature of CGBVS is critically different from existing predictive methods, such as LBVS, which depend on similarities between test and reference ligands, and focus on a single protein or highly homologous proteins. In particular, CGBVS is useful for targets with undefined ligands because this method can use CPIs with target proteins that exhibit lower levels of homology.
In summary, we have demonstrated that data mining of multiple CPIs is of great practical value for exploration of chemical space. As a predictive model, CGBVS could provide an important step in the discovery of such multi-target drugs by identifying the group of proteins targeted by a particular ligand, leading to innovation in pharmaceutical research.
The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold-hopping compounds. Through a machine-learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G-protein-coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand-screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.
PMCID: PMC3094066  PMID: 21364574
chemical genomics; data mining; drug discovery; ligand screening; systems chemical biology
3.  Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole 
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
The authors screen for compounds that show synergistic antifungal activity when combined with the widely-used fungistatic drug fluconazole. Chemogenomic profiling explains the mode of action of synergistic drugs and allows the prediction of additional drug synergies.
Chemical screens with a library enriched for known drugs identified a diverse set of 148 compounds that potentiated the action of the antifungal drug fluconazole against the fungal pathogens Cryptococcus neoformans, Cryptococcus gattii and Candida albicans, and the model yeast Saccharomyces cerevisiae, often in a species-specific manner.Chemogenomic profiles of six confirmed hits in S. cerevisiae revealed different modes of action and enabled the prediction of additional synergistic combinations; three-way synergistic interactions exhibited even stronger synergies at low doses of fluconazole.The synergistic combination of fluconazole and the antidepressant sertraline was active against fluconazole-resistant clinical fungal isolates and in an in vivo model of Cryptococcal infection.
Rising fungal infection rates, especially among immune-suppressed individuals, represent a serious clinical challenge (Gullo, 2009). Cancer, organ transplant and HIV patients, for example, often succumb to opportunistic fungal pathogens. The limited repertoire of approved antifungal agents and emerging drug resistance in the clinic further complicate the effective treatment of systemic fungal infections. At the molecular level, the paucity of fungal-specific essential targets arises from the conserved nature of cellular functions from yeast to humans, as well as from the fact that many essential yeast genes can confer viability at a fraction of wild-type dosage (Yan et al, 2009). Although only ∼1100 of the ∼6000 genes in yeast are essential, almost all genes become essential in specific genetic backgrounds in which another non-essential gene has been deleted or otherwise attenuated, an effect termed synthetic lethality (Tong et al, 2001). Genome-scale surveys suggest that over 200 000 binary synthetic lethal gene combinations dominate the yeast genetic landscape (Costanzo et al, 2010). The genetic buffering phenomenon is also manifest as a plethora of differential chemical–genetic interactions in the presence of sublethal doses of bioactive compounds (Hillenmeyer et al, 2008). These observations frame the difficulty of interdicting network functions in eukaryotic pathogens with single agent therapeutics. At the same time, however, this genetic network organization suggests that judicious combinations of small molecule inhibitors of both essential and non-essential targets may elicit additive or synergistic effects on cell growth (Sharom et al, 2004; Lehar et al, 2008). Unbiased screens for drugs that synergistically enhance a specific bioactive effect, but which are not themselves individually active—termed a syncretic combination—are one means to substantially elaborate chemical space (Keith et al, 2005). Indeed, compounds that enhance the activity of known agents in model yeast and cancer cell line systems have been identified both by focused small molecule library screens and by computational methods (Borisy et al, 2003; Lehar et al, 2007; Nelander et al, 2008; Jansen et al, 2009; Zinner et al, 2009).
To extend the stratagem of chemical synthetic lethality to clinically relevant fungal pathogens, we screened a bioactive library of known drugs for synergistic enhancers of the widely used fungistatic drug fluconazole against the clinically relevant pathogens C. albicans, C. neoformans and C. gattii, as well as the genetically tractable budding yeast S. cerevisiae. Fluconazole is an azole drug that inhibits lanosterol 14α-demethylase, the gene product of ERG11, an essential cytochrome P450 enzyme in the ergosterol biosynthetic pathway (Groll et al, 1998). We identified 148 drugs that potentiate the antifungal action of fluconazole against the four species. These syncretic compounds had not been previously recognized in the clinic as antifungal agents, and many acted in a species-specific manner, often in a potent fungicidal manner.
To understand the mechanisms of synergism, we interrogated six syncretic drugs—trifluoperazine, tamoxifen, clomiphene, sertraline, suloctidil and L-cycloserine—in genome-wide chemogenomic profiles of the S. cerevisiae deletion strain collection (Giaever et al, 1999). These profiles revealed that membrane, vesicle trafficking and lipid biosynthesis pathways are targeted by five of the synergizers, whereas the sphingolipid biosynthesis pathway is targeted by L-cycloserine. Cell biological assays confirmed the predicted membrane disruption effects of the former group of compounds, which may perturb ergosterol metabolism, impair fluconazole export by drug efflux pumps and/or affect active import of fluconazole (Kuo et al, 2010; Mansfield et al, 2010). Based on the integration of chemical–genetic and genetic interaction space, a signature set of deletion strains that are sensitive to the membrane active synergizers correctly predicted additional drug synergies with fluconazole. Similarly, the L-cycloserine chemogenomic profile correctly predicted a synergistic interaction between fluconazole and myriocin, another inhibitor of sphingolipid biosynthesis. The structure of genetic networks suggests that it should be possible to devise higher order drug combinations with even greater selectivity and potency (Sharom et al, 2004). In an initial test of this concept, we found that the combination of a non-synergistic pair drawn from the membrane active and sphingolipid target classes exhibited potent three-way synergism with a low dose of fluconazole. Finally, the combination of sertraline and fluconazole was active in a G. mellonella model of Cryptococcal infection, and was also efficacious against fluconazole-resistant clinical isolates of C. albicans and C. glabrata.
Collectively, these results demonstrate that the combinatorial redeployment of known drugs defines a powerful antifungal strategy and establish a number of potential lead combinations for future clinical assessment.
Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off-patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species- or genus-specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole-resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.
PMCID: PMC3159983  PMID: 21694716
antifungal; combination; pathogen; resistance; synergism
4.  Designing Focused Chemical Libraries Enriched in Protein-Protein Interaction Inhibitors using Machine-Learning Methods 
PLoS Computational Biology  2010;6(3):e1000695.
Protein-protein interactions (PPIs) may represent one of the next major classes of therapeutic targets. So far, only a minute fraction of the estimated 650,000 PPIs that comprise the human interactome are known with a tiny number of complexes being drugged. Such intricate biological systems cannot be cost-efficiently tackled using conventional high-throughput screening methods. Rather, time has come for designing new strategies that will maximize the chance for hit identification through a rationalization of the PPI inhibitor chemical space and the design of PPI-focused compound libraries (global or target-specific). Here, we train machine-learning-based models, mainly decision trees, using a dataset of known PPI inhibitors and of regular drugs in order to determine a global physico-chemical profile for putative PPI inhibitors. This statistical analysis unravels two important molecular descriptors for PPI inhibitors characterizing specific molecular shapes and the presence of a privileged number of aromatic bonds. The best model has been transposed into a computer program, PPI-HitProfiler, that can output from any drug-like compound collection a focused chemical library enriched in putative PPI inhibitors. Our PPI inhibitor profiler is challenged on the experimental screening results of 11 different PPIs among which the p53/MDM2 interaction screened within our own CDithem platform, that in addition to the validation of our concept led to the identification of 4 novel p53/MDM2 inhibitors. Collectively, our tool shows a robust behavior on the 11 experimental datasets by correctly profiling 70% of the experimentally identified hits while removing 52% of the inactive compounds from the initial compound collections. We strongly believe that this new tool can be used as a global PPI inhibitor profiler prior to screening assays to reduce the size of the compound collections to be experimentally screened while keeping most of the true PPI inhibitors. PPI-HitProfiler is freely available on request from our CDithem platform website,
Author Summary
Protein-protein interactions (PPIs) are essential to life and various diseases states are associated with aberrant PPIs. Therefore significant efforts are dedicated to this new class of therapeutic targets. Even though it might not be possible to modulate the estimated 650,000 PPIs that regulate human life with drug-like compounds, a sizeable number of PPI should be druggable. Only 10-15% of the human genome is thought to be druggable with around 1000-3000 druggable protein targets. A hypothetical similar ratio for PPIs would bring the number of druggable PPIs to about 65,000, although no data can yet support such a hypothesis. PPI have been historically intricate to tackle with standard experimental and virtual screening techniques, possibly because of the shift in the chemical space between today's chemical libraries and PPI physico-chemical requirements. Therefore, one possible avenue to circumvent this conundrum is to design focused libraries enriched in putative PPI inhibitors. Here, we show how chemoinformatics can assist library design by learning physico-chemical rules from a data set of known PPI inhibitors and their comparison with regular drugs. Our study shows the importance of specific molecular shapes and a privileged number of aromatic bonds.
PMCID: PMC2832677  PMID: 20221258
5.  Model System for High-Throughput Screening of Novel Human Immunodeficiency Virus Protease Inhibitors in Escherichia coli 
Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS. To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective Escherichia coli-based assay system. This E. coli-based system involves coexpression of an engineered β-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the transframe region and the protease domain. Autoprocessing of the HIV protease precursor releases the mature HIV protease. Subsequently, the HIV protease cleaves β-galactosidase, resulting in a loss of the β-galactosidase activity, which can be detected in high-throughput screens. Using Food and Drug Administration-approved HIV protease inhibitors, this E. coli-based system is validated as a surrogate screening system for identifying inhibitors that not only possess inhibitory activity against HIV protease but also have solubility and permeability for in vivo activity. The usefulness of the E. coli-based system was demonstrated with the identification of a novel HIV protease inhibitor from a library of compounds that were prepared by an amide-forming reaction with transition-state analog cores. A novel inhibitor with a sulfonamide core of amprenavir, E2, has shown good correlation with the in vitro enzymatic assay and in vivo E. coli-based system. This system can also be used to generate drug resistance profiles that could be used to suggest therapeutic uses of HIV protease inhibitors to treat the drug-resistant HIV strains. This simple yet efficient E. coli system not only represents a screening platform for high-throughput identification of leads targeting the HIV proteases but also can be adapted to all other classes of proteases.
PMCID: PMC434161  PMID: 15215092
6.  High Content Image-Based Screening of a Protease Inhibitor Library Reveals Compounds Broadly Active against Rift Valley Fever Virus and Other Highly Pathogenic RNA Viruses 
High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology.
Author Summary
Rift Valley fever (RVF) is an arthropod-borne viral zoonosis that occurs in large parts of sub-Saharan and North Africa and in 2000 emerged outside the African continent for the first time, raising concerns that it could further expand its geographical range. The disease in humans can result in encephalitis or hemorrhagic fever and in ruminants often results in abortion in pregnant females. Due to the lack of a licensed and commercially available vaccine, efforts to discover effective antiviral drugs are underway. Drug discovery using high content image-based screening is an effective tool that has been successfully used to identify new drugs. In this study, we developed an image-based assay to identify compounds active against RVF virus and other highly pathogenic human viruses. We demonstrated the usefulness of our image-based high content assay in identifying potential RVF antivirals by screening a small subset of chemical compounds for inhibition of RVF virus in a human cell line (HeLa) and partially characterized their mechanism of action within infected cells. The methods we developed in this study will be useful in discovering new effective drugs to combat Rift Valley fever.
PMCID: PMC4140764  PMID: 25144302
7.  High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK3:CYC6 Cyclin-Dependent Kinase 
Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.
Methodology/Principal Findings
In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.
The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.
Author Summary
CRK3, a cdc2-related serine/threonine protein kinase of the CDK family, is essential for transition through the G2-M phase checkpoint of the Leishmania cell cycle. An expression and purification system has been developed to produce active L. major CRK3 in complex with a cyclin partner, CYC6. CRK3:CYC6 was used to develop an assay suitable for high throughput screening (HTS) using IMAP fluorescence polarization technology. Two compound chemical libraries were screened against CRK3:CYC6 and counter screened against a human cyclin-dependent kinase complex CDK2:CycA. Two main chemical families of inhibitors were identified that specifically inhibited the leishmanial cyclin-dependent kinase, the azapurines and the thiazoles. Structure activity relationship (SAR) analysis of the hits identified the chemical groups attached to the azapurine scaffold that are essential for the inhibition of CRK3:CYC6 protein kinase activity. The CRK3:CYC6 hits were subsequently tested against a panel of 11 mammalian kinases including human CDK1:CYCB, human CDK2:CYCA and human CDK4:CYCD1 to determine their selectivity. Compounds selective to CRK3:CYC6 were tested against Leishmania. Progress towards synthesising potent and selective derivatives of the HTS hits are discussed, with the view to evaluating their potential for the development of novel therapeutics against leishmaniasis.
PMCID: PMC3071374  PMID: 21483720
8.  High-Throughput Screening (HTS) and Hit Validation to Identify Small Molecule Inhibitors with Activity against NS3/4A proteases from Multiple Hepatitis C Virus Genotypes 
PLoS ONE  2013;8(10):e75144.
Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC50 value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.
PMCID: PMC3793977  PMID: 24130685
9.  Selective Inhibitors of Protozoan Protein N-myristoyltransferases as Starting Points for Tropical Disease Medicinal Chemistry Programs 
Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (Hs1 and Hs2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. Analysis of the screening results has shown that structure-activity relationships (SAR) for Leishmania NMT are divergent from all other NMTs tested, a finding not predicted by sequence similarity calculations, resulting in the identification of four novel series of Leishmania-selective NMT inhibitors. We found a strong overlap between the SARs for Plasmodium NMT and both human NMTs, suggesting that achieving an appropriate selectivity profile will be more challenging. However, we did discover two novel series with selectivity for Plasmodium NMT over the other NMT orthologues in this study, and an additional two structurally distinct series with selectivity over Leishmania NMT. We believe that release of results from this study into the public domain will accelerate the discovery of NMT inhibitors to treat malaria and leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as Medical Research Council and Wellcome Trust can stimulate research for neglected diseases.
Author Summary
Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (HsNMT1 and HsNMT2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. We have identified eight series of protozoan NMT inhibitors, six having good selectivity for either Plasmodium or Leishmania NMTs over the other orthologues in this study. We believe that all of these series could form the basis of medicinal chemistry programs to deliver drug candidates against either malaria or leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as the UK Medical Research Council and Wellcome Trust can stimulate research for neglected diseases.
PMCID: PMC3335879  PMID: 22545171
10.  Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes 
We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.
Author Summary
We have developed an assay for screening test compounds for their ability to kill intracellular amastigotes of Leishmania parasites, causative agents of human leishmaniasis. The assay is based on freeing amastigotes from infected macrophages by mild detergent lysis and measuring the number of parasites following their extracellular replication by a fluorescence-based method. The validity of the assay has been confirmed using the gold standard drugs, Amphotericin B and Miltefosine, which kill parasites at highly reproducible concentrations. Our results show that this assay is easily transferable between laboratories, can be adapted to specific applications and used to test any parasite species or strain, and does not rely on genetically-modified parasites. These features will enable its use in screening isolates taken directly from patients, vectors or reservoir hosts. We used this assay, in parallel with enzyme activity data, to test lead-like and hit-like inhibitors of a validated target enzyme, Leishmania N-myristoyltransferase (NMT). Compounds from two of four newly-identified Leishmania NMT-selective hit series displayed host cell cytotoxicity, while all four series displayed low translation of enzyme to cellular activity in analysis of intracellular parasite viability. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.
PMCID: PMC4270692  PMID: 25522361
11.  A comparative study of fragment screening methods on the p38α kinase: new methods, new insights 
The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore™ T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE™) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.
Electronic supplementary material
The online version of this article (doi:10.1007/s10822-011-9454-9) contains supplementary material, which is available to authorized users.
PMCID: PMC3155752  PMID: 21732248
p38α; Surface-plasmon resonance; Fragment screening; X-ray crystallography
12.  Assay Strategies for the Discovery and Validation of Therapeutics Targeting Brugia pahangi Hsp90 
The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target.
Author Summary
Helminth diseases of humans remain a major problem in many parts of the tropics. Treatment of these parasitic infections is restricted to a limited number of drugs and few new compounds are in development. One of the major obstacles to the development of new therapeutics is the lack of high-throughput screens that can be adapted to parasitic species for the identification of small molecule inhibitors. Here we present a simple, inexpensive assay for the identification of inhibitors of Hsp90 in filarial worms. The assay, first described for the identification of Hsp90 inhibitors in tumor cells, does not require recombinant protein but relies upon the ability of a fluorescently labelled drug to bind to Hsp90 in the context of a soluble fraction of worm homogenate. We validated the assay using known inhibitors of Hsp90, including derivatives of the synthetic purine-scaffold series of Hsp90 inhibitors and were able to show a differential sensitivity to these compounds between human and Brugia Hsp90.
PMCID: PMC2886105  PMID: 20559560
13.  Cellular Ser/Thr-Kinase Assays Using Generic Peptide Substrates 
High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves a) coexpression of targeted kinases with promiscuous peptide substrates and b) quantification of intracellular substrate phosphorylation by homogeneous TR-FRET. Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2δ assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC50-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.
PMCID: PMC2774688  PMID: 20161828
14.  Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds 
Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC50s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC50s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC50s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.
PMCID: PMC3910886  PMID: 24277038
15.  The Activities of Current Antimalarial Drugs on the Life Cycle Stages of Plasmodium: A Comparative Study with Human and Rodent Parasites 
PLoS Medicine  2012;9(2):e1001169.
Michael Delves and colleagues compare the activity of 50 current and experimental antimalarials against liver, sexual blood, and mosquito stages of selected human and nonhuman parasite species, including Plasmodium falciparum, Plasmodium berghei, and Plasmodium yoelii.
Malaria remains a disease of devastating global impact, killing more than 800,000 people every year—the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance.
Methods and Findings
Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony.
These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle–wide analyses of drugs for other pathogens with complex life cycles.
Please see later in the article for the Editors' Summary
Editors' Summary
Malaria is a life-threatening disease caused by the Plasmodium parasite, which is transmitted to people through the bites of infected mosquitoes. According to latest global estimates, about 250 million people are infected with malaria every year with roughly 800,000 deaths—most occurring among young children living in Africa. Malaria also causes severe morbidity in children, such as anemia, low birth weight, and neurological problems, which compromise the health and development of millions of children living in malaria endemic areas. In addition to strategies that scale up and roll out the prevention of malaria, such as country-wide programs to provide insecticide-treating bednets, in the goal to eradicate malaria, the global health community has refocused efforts on the treatment of malaria, including finding new compounds that target different stages of the parasite life cycle as it passes from vector to host and back.
The interruption of malaria transmission worldwide is one of the greatest challenges for the global health community. In January 2011, this journal published a series on The Malaria Eradication Research Agenda (malERA), which described a set of research and development priorities, identified key knowledge gaps and the necessary tools needed, and introduced a draft research and development agenda for the worldwide eradication of malaria.
Why Was This Study Done?
Most currently available antimalarial drugs primarily target the disease-causing parasites' stages in the human blood system. But to eradicate malaria, new drugs that block transmission of the parasite between the human host and the mosquito vector, and eliminate the various stages of the parasite during its cycle in the human body, are needed. In this laboratory study, the researchers compared the profiles of all available and experimental antimalarials and analyzed each drug for activity against each specific stage in the malaria parasite's life cycle to provide a reference set of methods and data, that might serve as a benchmark to help guide the malaria research community in assessing the potential of newly discovered antimalarials. Furthermore, this analysis could provide insights into which chemical drug classes might provide transmission-blocking capabilities—an essential component of malaria eradication.
What Did the Researchers Do and Find?
The researchers used novel laboratory techniques under standardized conditions to develop a series of novel assays to analyze the activities of 50 antimalarial compounds (current drugs and those under development) against three Plasmodium species encompassing every major cellular strategy of the malarial life cycle including drug resistant parasite strains. In their comparative analysis, the researchers undertook a chemical profiling approach to identify the drugs that block transmission from the host to the mosquito vector and additionally suppress transmission from the mosquito to the human host.
The researchers highlighted some encouraging results; for example, the potencies of some antimalarials against the asexual blood stage of cultivated P. falciparum and P. vivax isolates show a very good correlation, suggesting that most of the pathways inhibited by antimalarials in P. falciparum may also be valid targets in P. vivax. The researchers also have shown that approved drugs, such as pyronaridine and atovaquone, can target liver and sexual stages in addition to asexual blood stages. Furthermore, the researchers found promising results for new compounds currently in clinical trials, such as the endoperoxide OZ439, a stable synthetic molecule currently being studied in a phase IIa clinical trial, which seemed to be a strong inhibitor of gametocyte maturation and gamete formation. The new 8-aminoquinoline, NPC-1161B, also inhibited sporogony.
What Do These Findings Mean?
The results of this analysis provide a valuable guide to help researchers decide which drugs and compounds show most promise as potential future antimalarial drugs for blocking the transmission of malaria. This study could also help researchers make decisions about which molecules could be best combined to provide the next generation of drugs that will succeed artemisinin compound therapy and support the eradication of malaria. Furthermore, this comprehensive approach to drug discovery could be applied to test drugs against other pathogens with complex life cycles.
Additional Information
Please access these Web sites via the online version of this summary at
The malERA a research agenda for malaria eradication sponsored collection, published by PLoS in January 2011, comprises 12 Review articles that discuss agendas in malaria research and development
PMCID: PMC3283556  PMID: 22363211
16.  Antibiotic Activity and Characterization of BB-3497, a Novel Peptide Deformylase Inhibitor 
Peptide deformylase (PDF) is an essential bacterial metalloenzyme which deformylates the N-formylmethionine of newly synthesized polypeptides and as such represents a novel target for antibacterial chemotherapy. To identify novel PDF inhibitors, we screened a metalloenzyme inhibitor library and identified an N-formyl-hydroxylamine derivative, BB-3497, and a related natural hydroxamic acid antibiotic, actinonin, as potent and selective inhibitors of PDF. To elucidate the interactions that contribute to the binding affinity of these inhibitors, we determined the crystal structures of BB-3497 and actinonin bound to Escherichia coli PDF at resolutions of 2.1 and 1.75 Å, respectively. In both complexes, the active-site metal atom was pentacoordinated by the side chains of Cys 90, His 132, and His 136 and the two oxygen atoms of N-formyl-hydroxylamine or hydroxamate. BB-3497 had activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, and activity against some gram-negative bacteria. Time-kill analysis showed that the mode of action of BB-3497 was primarily bacteriostatic. The mechanism of resistance was via mutations within the formyltransferase gene, as previously described for actinonin. While actinonin and its derivatives have not been used clinically because of their poor pharmacokinetic properties, BB-3497 was shown to be orally bioavailable. A single oral dose of BB-3497 given 1 h after intraperitoneal injection of S. aureus Smith or methicillin-resistant S. aureus protected mice from infection with median effective doses of 8 and 14 mg/kg of body weight, respectively. These data validate PDF as a novel target for the design of a new generation of antibacterial agents.
PMCID: PMC90327  PMID: 11158755
17.  A functional proteomics screen of proteases in colorectal carcinoma. 
Molecular Medicine  2000;6(5):450-460.
BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. RESULTS: The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. CONCLUSIONS: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.
PMCID: PMC1949953  PMID: 10952024
18.  Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics 
Journal of Clinical Investigation  2004;114(8):1107-1116.
Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics.
PMCID: PMC522256  PMID: 15489958
19.  Pathway-based Screening Strategy for Multitarget Inhibitors of Diverse Proteins in Metabolic Pathways 
PLoS Computational Biology  2013;9(7):e1003127.
Many virtual screening methods have been developed for identifying single-target inhibitors based on the strategy of “one–disease, one–target, one–drug”. The hit rates of these methods are often low because they cannot capture the features that play key roles in the biological functions of the target protein. Furthermore, single-target inhibitors are often susceptible to drug resistance and are ineffective for complex diseases such as cancers. Therefore, a new strategy is required for enriching the hit rate and identifying multitarget inhibitors. To address these issues, we propose the pathway-based screening strategy (called PathSiMMap) to derive binding mechanisms for increasing the hit rate and discovering multitarget inhibitors using site-moiety maps. This strategy simultaneously screens multiple target proteins in the same pathway; these proteins bind intermediates with common substructures. These proteins possess similar conserved binding environments (pathway anchors) when the product of one protein is the substrate of the next protein in the pathway despite their low sequence identity and structure similarity. We successfully discovered two multitarget inhibitors with IC50 of <10 µM for shikimate dehydrogenase and shikimate kinase in the shikimate pathway of Helicobacter pylori. Furthermore, we found two selective inhibitors (IC50 of <10 µM) for shikimate dehydrogenase using the specific anchors derived by our method. Our experimental results reveal that this strategy can enhance the hit rates and the pathway anchors are highly conserved and important for biological functions. We believe that our strategy provides a great value for elucidating protein binding mechanisms and discovering multitarget inhibitors.
Author Summary
Many drug development strategies focus on designing inhibitors for single targets. These inhibitors often lose potency owing to mutations in the protein binding sites and are ineffective for complex diseases. Multitarget inhibitors can decrease probability of drug resistance and enhance the therapeutic efficiency; however, identifying them is still a challenge because targets often have low sequence and structure similarities in their binding sites. Here we propose a pathway-based screening strategy that simultaneously screens proteins in a metabolic pathway for discovering multitarget inhibitors. Because these proteins interact with similar metabolites and modify them step-by-step, the proteins share similarities in binding sites. We developed pathway site-moiety maps that present the conserved binding environments of the proteins without relying on the sequence or structure alignment. Compounds that bind these conserved binding environments are often multitarget inhibitors. We applied this strategy to the shikimate pathway of Helicobacter pylori, and discovered two multitarget inhibitors (IC50<10 µM) for shikimate dehydrogenase and shikimate kinase. In addition, we found two selective inhibitors based on specific binding environments for shikimate dehydrogenase. Thus the pathway-based screening strategy is useful for identifying multitarget inhibitors and elucidating protein-ligand binding mechanisms and has the potential to be applied to human diseases.
PMCID: PMC3701698  PMID: 23861662
20.  Proteomic profiling of metalloprotease activities with cocktails of active-site probes 
Nature chemical biology  2006;2(5):274-281.
Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a ‘cocktail’ to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.
PMCID: PMC1538544  PMID: 16565715
21.  Bifunctional Role of the Treponema pallidum Extracellular Matrix Binding Adhesin Tp0751 ▿  
Infection and Immunity  2010;79(3):1386-1398.
Treponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. The T. pallidum adhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction of T. pallidum with host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, and in vitro degradation assays confirmed that recombinant Tp0751 purified from both insect and Escherichia coli expression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of a T. pallidum protease capable of degrading host components and thus provides novel insight into the mechanism of T. pallidum dissemination.
PMCID: PMC3067502  PMID: 21149586
22.  Comparison of the Cancer Gene Targeting and Biochemical Selectivities of All Targeted Kinase Inhibitors Approved for Clinical Use 
PLoS ONE  2014;9(3):e92146.
The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013), and for six of these drugs, the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers, suggesting that cancers dependent on mutant CTNNB1 will respond to trametinib and other MEK inhibitors, and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR, ABL1 and BRAF(V600E)-driven cell growth, and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors, we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action.
PMCID: PMC3961306  PMID: 24651269
23.  Characterization of the Zinc-Containing Metalloprotease Encoded by zpx and Development of a Species-Specific Detection Method for Enterobacter sakazakii▿  
Applied and Environmental Microbiology  2007;73(13):4142-4151.
Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused “rounding” of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37°C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.
PMCID: PMC1932767  PMID: 17483271
24.  The Molecular Basis of Drug Resistance against Hepatitis C Virus NS3/4A Protease Inhibitors 
PLoS Pathogens  2012;8(7):e1002832.
Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.
Author Summary
Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. New classes of directly-acting antiviral agents that target various HCV enzymes are being developed. Two such drugs that target the essential HCV NS3/4A protease are approved by the FDA and several others are at various stages of clinical development. These drugs, when used in combination with pegylated interferon and ribavirin, significantly improve treatment outcomes. However HCV evolves very quickly and drug resistance develops against directly-acting antiviral agents. Thus, despite the therapeutic success of NS3/4A protease inhibitors, their long-term effectiveness is challenged by drug resistance. Our study explains in atomic detail how and why drug resistance occurs for four chemically representative protease inhibitors –telaprevir, danoprevir, vaniprevir and MK-5172. Potentially with this knowledge, new drugs could be developed that are less susceptible to drug resistance. More generally, understanding the underlying mechanisms by which drug resistance occurs can be incorporated in drug development to many quickly evolving diseases.
PMCID: PMC3406087  PMID: 22910833
25.  Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer 
Executive Summary
In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.
Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenetics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.
The following reports can be publicly accessed at the MAS website at: or at
Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer: An Evidence-Based Analysis
Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based Analysis
K-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based Analysis
The Medical Advisory Secretariat undertook a systematic review of the evidence on the clinical effectiveness and cost-effectiveness of epidermal growth factor receptor (EGFR) mutation testing compared with no EGFR mutation testing to predict response to tyrosine kinase inhibitors (TKIs), gefitinib (Iressa®) or erlotinib (Tarceva®) in patients with advanced non-small cell lung cancer (NSCLC).
Clinical Need: Target Population and Condition
With an estimated 7,800 new cases and 7,000 deaths last year, lung cancer is the leading cause of cancer deaths in Ontario. Those with unresectable or advanced disease are commonly treated with concurrent chemoradiation or platinum-based combination chemotherapy. Although response rates to cytotoxic chemotherapy for advanced NSCLC are approximately 30 to 40%, all patients eventually develop resistance and have a median survival of only 8 to 10 months. Treatment for refractory or relapsed disease includes single-agent treatment with docetaxel, pemetrexed or EGFR-targeting TKIs (gefitinib, erlotinib). TKIs disrupt EGFR signaling by competing with adenosine triphosphate (ATP) for the binding sites at the tyrosine kinase (TK) domain, thus inhibiting the phosphorylation and activation of EGFRs and the downstream signaling network. Gefitinib and erlotinib have been shown to be either non-inferior or superior to chemotherapy in the first- or second-line setting (gefitinib), or superior to placebo in the second- or third-line setting (erlotinib).
Certain patient characteristics (adenocarcinoma, non-smoking history, Asian ethnicity, female gender) predict for better survival benefit and response to therapy with TKIs. In addition, the current body of evidence shows that somatic mutations in the EGFR gene are the most robust biomarkers for EGFR-targeting therapy selection. Drugs used in this therapy, however, can be costly, up to C$ 2000 to C$ 3000 per month, and they have only approximately a 10% chance of benefiting unselected patients. For these reasons, the predictive value of EGFR mutation testing for TKIs in patients with advanced NSCLC needs to be determined.
The Technology: EGFR mutation testing
The EGFR gene sequencing by polymerase chain reaction (PCR) assays is the most widely used method for EGFR mutation testing. PCR assays can be performed at pathology laboratories across Ontario. According to experts in the province, sequencing is not currently done in Ontario due to lack of adequate measurement sensitivity. A variety of new methods have been introduced to increase the measurement sensitivity of the mutation assay. Some technologies such as single-stranded conformational polymorphism, denaturing high-performance liquid chromatography, and high-resolution melting analysis have the advantage of facilitating rapid mutation screening of large numbers of samples with high measurement sensitivity but require direct sequencing to confirm the identity of the detected mutations. Other techniques have been developed for the simple, but highly sensitive detection of specific EGFR mutations, such as the amplification refractory mutations system (ARMS) and the peptide nucleic acid-locked PCR clamping. Others selectively digest wild-type DNA templates with restriction endonucleases to enrich mutant alleles by PCR. Experts in the province of Ontario have commented that currently PCR fragment analysis for deletion and point mutation conducts in Ontario, with measurement sensitivity of 1% to 5%.
Research Questions
In patients with locally-advanced or metastatic NSCLC, what is the clinical effectiveness of EGFR mutation testing for prediction of response to treatment with TKIs (gefitinib, erlotinib) in terms of progression-free survival (PFS), objective response rates (ORR), overall survival (OS), and quality of life (QoL)?
What is the impact of EGFR mutation testing on overall clinical decision-making for patients with advanced or metastatic NSCLC?
What is the cost-effectiveness of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the first-line setting?
What is the budget impact of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the second- or third-line setting?
A literature search was performed on March 9, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, OVID EMBASE, Wiley Cochrane, CINAHL, Centre for Reviews and Dissemination/International Agency for Health Technology Assessment for studies published from January 1, 2004 until February 28, 2010 using the following terms:
Non-Small-Cell Lung Carcinoma
Epidermal Growth Factor Receptor
An automatic literature update program also extracted all papers published from February 2010 until August 2010. Abstracts were reviewed by a single reviewer and for those studies meeting the eligibility criteria full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with unknown eligibility were reviewed with a second clinical epidemiologist, and then a group of epidemiologists, until consensus was established. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology.
The inclusion criteria were as follows:
Population: patients with locally advanced or metastatic NSCLC (stage IIIB or IV)
Procedure: EGFR mutation testing before treatment with gefitinib or erlotinib
Language: publication in English
Published health technology assessments, guidelines, and peer-reviewed literature (abstracts, full text, conference abstract)
Outcomes: progression-free survival (PFS), Objective response rate (ORR), overall survival (OS), quality of life (QoL).
The exclusion criteria were as follows:
Studies lacking outcomes specific to those of interest
Studies focused on erlotinib maintenance therapy
Studies focused on gefitinib or erlotinib use in combination with cytotoxic agents or any other drug
Grey literature, where relevant, was also reviewed.
Outcomes of Interest
ORR determined by means of the Response Evaluation Criteria in Solid Tumours (RECIST)
Quality of Evidence
The quality of the Phase II trials and observational studies was based on the method of subject recruitment and sampling, possibility of selection bias, and generalizability to the source population. The overall quality of evidence was assessed as high, moderate, low or very low according to the GRADE Working Group criteria.
Summary of Findings
Since the last published health technology assessment by Blue Cross Blue Shield Association in 2007 there have been a number of phase III trials which provide evidence of predictive value of EGFR mutation testing in patients who were treated with gefitinib compared to chemotherapy in the first- or second-line setting. The Iressa Pan Asian Study (IPASS) trial showed the superiority of gefitinib in terms of PFS in patients with EGFR mutations versus patients with wild-type EGFR (Hazard ratio [HR], 0.48, 95%CI; 0.36-0.64 versus HR, 2.85; 95%CI, 2.05-3.98). Moreover, there was a statistically significant increased ORR in patients who received gefitinib and had EGFR mutations compared to patients with wild-type EGFR (71% versus 1%). The First-SIGNAL trial in patients with similar clinical characteristics as IPASS as well as the NEJ002 and WJTOG3405 trials that included only patients with EGFR mutations, provide confirmation that gefitinib is superior to chemotherapy in terms of improved PFS or higher ORR in patients with EGFR mutations. The INTEREST trial further indicated that patients with EGFR mutations had prolonged PFS and higher ORR when treated with gefitinib compared with docetaxel.
In contrast, there is still a paucity of strong evidence regarding the predictive value of EGFR mutation testing for response to erlotinib in the second- or third-line setting. The BR.21 trial randomized 731 patients with NSCLC who were refractory or intolerant to prior first- or second-line chemotherapy to receive erlotinib or placebo. While the HR of 0.61 (95%CI, 0.51-0.74) favored erlotinib in the overall population, this was not a significant in the subsequent retrospective subgroup analysis. A retrospective evaluation of 116 of the BR.21 tumor samples demonstrated that patients with EGFR mutations had significantly higher ORRs when treated with erlotinib compared with placebo (27% versus 7%; P=0.03). However, erlotinib did not confer a significant survival benefit compared with placebo in patients with EGFR mutations (HR, 0.55; 95%CI, 0.25-1.19) versus wild-type (HR, 0.74; 95%CI, 0.52-1.05). The interaction between EGFR mutation status and erlotinib use was not significant (P=0.47). The lack of significance could be attributable to a type II error since there was a low sample size that was available for subgroup analysis.
A series of phase II studies have examined the clinical effectiveness of erlotinib in patients known to have EGFR mutations. Evidence from these studies has consistently shown that erlotinib yields a very high ORR (typically 70% vs. 4%) and a prolonged PFS (9 months vs. 2 months) in patients with EGFR mutations compared with patients with wild-type EGFR. Although having a prolonged PFS and higher respond in EGFR mutated patients might be due to a better prognostic profile regardless of the treatment received. In the absence of a comparative treatment or placebo control group, it is difficult to determine if the observed differences in survival benefit in patients with EGFR mutation is attributed to prognostic or predictive value of EGFR mutation status.
Based on moderate quality of evidence, patients with locally advanced or metastatic NSCLC with adenocarcinoma histology being treated with gefitinib in the first-line setting are highly likely to benefit from gefitinib if they have EGFR mutations compared to those with wild-type EGFR. This advantage is reflected in improved PFS, ORR and QoL in patients with EGFR mutation who are being treated with gefitinib relative to patients treated with chemotherapy.
Based on low quality of evidence, in patients with locally advanced or metastatic NSCLC who are being treated with erlotinib, the identification of EGFR mutation status selects those who are most likely to benefit from erlotinib relative to patients treated with placebo in the second or third-line setting.
PMCID: PMC3377519  PMID: 23074402

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