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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.  New tools for dissecting protease function: implications for inhibitor design, drug discovery and probe development 
Proteases have long been considered targets for pharmaceutical development because of our deep understanding of their enzymatic mechanism and their regulatory roles in many pathologies. However, despite our ability to develop potent inhibitors, many clinical lead compounds have failed due either to a lack of specificity or a limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to first understand protease function and second, to expand the platform of inhibitor development beyond active site-directed design and in vitro optimization. Several newly developed technologies will enable much broader assessment of drug selectivity in living cells and in animal models, allowing for lead optimization to focus on compounds that show high specificity and minimal side effects in vivo. In this perspective, we highlight the current advances in the development of new chemical probes, proteomic methods, and screening tools that we feel will help facilitate this paradigm shift in drug discovery methods.
PMCID: PMC3513415  PMID: 22218294
3.  Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity 
Nature Biotechnology  2011;29(11):1039-1045.
Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them.
PMCID: PMC3230241  PMID: 22037377
4.  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
5.  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
6.  Confirming Target Engagement for Reversible Inhibitors In Vivo by Kinetically Tuned Activity-Based Probes 
Journal of the American Chemical Society  2012;134(25):10345-10348.
The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.
PMCID: PMC3392194  PMID: 22690931
7.  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
8.  Molecular Basis for Drug Resistance in HIV-1 Protease 
Viruses  2010;2(11):2509-2535.
HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.
PMCID: PMC3185577  PMID: 21994628
drug resistance; HIV-1 protease; protease inhibitors; substrate envelope; structure based drug design
9.  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
10.  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
11.  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
12.  Novel Method for Probing the Specificity Binding Profile of Ligands: Applications to HIV Protease 
Chemical biology & drug design  2008;71(5):387-407.
A detailed understanding of factors influencing the binding specificity of a ligand to a set of desirable targets and undesirable decoys is a key step in the design of potent and selective therapeutics. We have developed a general method for optimizing binding specificity in ligand–receptor complexes based on the theory of electrostatic charge optimization. This methodology can be used to tune the binding of a ligand to a panel of potential targets and decoys, along the continuum from narrow binding to only one partner to broad binding to the entire panel. Using HIV-1 protease as a model system, we probe specificity in three distinct ways. First, we probe interactions that could make the promiscuous protease inhibitor pepstatin more selective toward HIV-1 protease. Next, we study clinically approved HIV-1 protease inhibitors and probe ways to broaden the binding profiles toward both wild-type HIV-1 protease and drug-resistant mutants. Finally, we study a conformational ensemble of wild-type HIV-1 protease to “design in” broad specificity to known drugs before resistance mutations arise. The results from this conformational ensemble were similar to those from the drug-resistant ensemble, suggesting the use of a conformational wild-type ensemble as a tool to develop escape-mutant resistant inhibitors.
PMCID: PMC3480184  PMID: 18384529
binding affinity; continuum electrostatics; charge optimization
13.  Applying Ligands Profiling Using Multiple Extended Electron Distribution Based Field Templates and Feature Trees Similarity Searching in the Discovery of New Generation of Urea-Based Antineoplastic Kinase Inhibitors 
PLoS ONE  2012;7(11):e49284.
This study provides a comprehensive computational procedure for the discovery of novel urea-based antineoplastic kinase inhibitors while focusing on diversification of both chemotype and selectivity pattern. It presents a systematic structural analysis of the different binding motifs of urea-based kinase inhibitors and the corresponding configurations of the kinase enzymes. The computational model depends on simultaneous application of two protocols. The first protocol applies multiple consecutive validated virtual screening filters including SMARTS, support vector-machine model (ROC = 0.98), Bayesian model (ROC = 0.86) and structure-based pharmacophore filters based on urea-based kinase inhibitors complexes retrieved from literature. This is followed by hits profiling against different extended electron distribution (XED) based field templates representing different kinase targets. The second protocol enables cancericidal activity verification by using the algorithm of feature trees (Ftrees) similarity searching against NCI database. Being a proof-of-concept study, this combined procedure was experimentally validated by its utilization in developing a novel series of urea-based derivatives of strong anticancer activity. This new series is based on 3-benzylbenzo[d]thiazol-2(3H)-one scaffold which has interesting chemical feasibility and wide diversification capability. Antineoplastic activity of this series was assayed in vitro against NCI 60 tumor-cell lines showing very strong inhibition of GI50 as low as 0.9 uM. Additionally, its mechanism was unleashed using KINEX™ protein kinase microarray-based small molecule inhibitor profiling platform and cell cycle analysis showing a peculiar selectivity pattern against Zap70, c-src, Mink1, csk and MeKK2 kinases. Interestingly, it showed activity on syk kinase confirming the recent studies finding of the high activity of diphenyl urea containing compounds against this kinase. Allover, the new series, which is based on a new kinase scaffold with interesting chemical diversification capabilities, showed that it exhibits its “emergent” properties by perturbing multiple unexplored kinase pathways.
PMCID: PMC3502486  PMID: 23185312
14.  Structure of Gentlyase, the neutral metalloprotease of Paenibacillus polymyxa  
The crystal structure of the metalloprotease Gentlyase is described and compared with the structures of other related thermolysin-like proteases.
Gentlyase is a bacterial extracellular metalloprotease that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases. The structure of thermolysin has been known since 1972 and that of Bacillus cereus neutral protease since 1992. However, the structure determination of other Bacillus neutral proteases has been hindered by their tendency to cannibalistic autolysis. High calcium conditions that allow the concentration and crystallization of the active Gentlyase metalloprotease without autoproteolysis were identified using thermal fluorescent shift assays. X-ray structures of the protease were solved in the absence and in the presence of the inhibitor phosphoramidon at 1.59 and 1.76 Å resolution, respectively. No domain movement was observed upon inhibitor binding, although such movement is thought to be a general feature of the thermolysin-like protease family. Further analysis of the structure shows that the observed calcium dependency of Gentlyase stability may arise from a partly degenerated calcium site Ca1–2 and a deletion near site Ca3.
PMCID: PMC3532130  PMID: 23275160
thermolysin; neutral proteases; calcium binding; Gentlyase
15.  Identification of exosite-targeting inhibitors of anthrax lethal factor by high throughput screening 
Chemistry & biology  2012;19(7):875-882.
Protease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small molecule library, and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases.
PMCID: PMC3408624  PMID: 22840775
16.  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
17.  Genotypic Testing for Human Immunodeficiency Virus Type 1 Drug Resistance 
Clinical Microbiology Reviews  2002;15(2):247-277.
There are 16 approved human immunodeficiency virus type 1 (HIV-1) drugs belonging to three mechanistic classes: protease inhibitors, nucleoside and nucleotide reverse transcriptase (RT) inhibitors, and nonnucleoside RT inhibitors. HIV-1 resistance to these drugs is caused by mutations in the protease and RT enzymes, the molecular targets of these drugs. Drug resistance mutations arise most often in treated individuals, resulting from selective drug pressure in the presence of incompletely suppressed virus replication. HIV-1 isolates with drug resistance mutations, however, may also be transmitted to newly infected individuals. Three expert panels have recommended that HIV-1 protease and RT susceptibility testing should be used to help select HIV drug therapy. Although genotypic testing is more complex than typical antimicrobial susceptibility tests, there is a rich literature supporting the prognostic value of HIV-1 protease and RT mutations. This review describes the genetic mechanisms of HIV-1 drug resistance and summarizes published data linking individual RT and protease mutations to in vitro and in vivo resistance to the currently available HIV drugs.
PMCID: PMC118066  PMID: 11932232
18.  In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases 
Chemistry & biology  2011;18(6):699-710.
Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against more than 200 kinases in native cell proteomes and reveal new biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected upon live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.
PMCID: PMC3142620  PMID: 21700206
19.  The Discovery of Novel 10,11-Dihydro-5H-dibenz[b,f]azepine SIRT2 Inhibitors 
MedChemComm  2012;10.1039/C2MD00290F.
Isoform selective inhibitors of the sirtuins (NAD+-dependent histone deacetylases) should enable an in depth study of the molecular biology underpinning these targets and how they are deregulated in diseases such as cancer and neurodegeneration. Herein, we present the discovery of structurally novel SIRT2 inhibitors. Hit molecule 8 was discovered through the chemical synthesis and biological characterization of a small-molecule compound library based around the 10,11-dihydro-5H-dibenz[b,f]azepine scaffold. In vitro screening assays revealed compound 8 to have an IC50 of 18 μM against SIRT2 and to exhibit more than 30-fold selectivity compared to SIRT1. Cellular assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed hit 8 to have antiproliferative activity at a concentration of 30 μM. Computational studies were performed to predict the SIRT2 binding mode and to rationalise the observed selectivity.
PMCID: PMC3856871  PMID: 24340169
20.  Substrate Inhibition Kinetic Model for West Nile Virus NS2B-NS3 Protease † 
Biochemistry  2008;47(45):11763-11770.
West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested peptide substrates could bind to two binding sites on the protease. Reaction product analogs also showed inhibition of the protease, demonstrating product inhibition in addition to, and distinct from, substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1′-P3′ interaction surface (i.e., the S1′-S3′ sites). Optimization of substrate analog inhibitors that target these two independent sites may lead to novel anti-WNV drugs.
PMCID: PMC2648976  PMID: 18855422
21.  The challenge of selecting protein kinase assays for lead discovery optimization 
Expert opinion on drug discovery  2008;3(6):607-621.
Protein kinases represent one of the most promising groups of drug targets owing to their involvement in such pathological conditions as cancer, inflammatory diseases, neural disorders, and metabolism problems. In the last few years, numerous pharmaceutical and biotech companies have established kinase high-throughput screening (HTS) programs, and the reagent and service industries for kinase assay platforms, kits, and profiling services have begun to thrive.
The plethora of different assay formats available today poses a great challenge to scientists who want to select a technology that will be cost efficient, convenient to use, and have low false positive and false negative rates.
In the current review, we summarize the most commonly used kinase assay methods in the drug discovery process, present the advantages and disadvantages of each of these methods, and discuss the challenges of discovering kinase inhibitors by using these technologies.
The decision of selecting the assay formats for HTS or service platform for profiling should take into account not only the final goals of the screens but also the limitation of resources.
PMCID: PMC2721227  PMID: 19662101
drug discovery; drug profiling; drug screening; ELISA; fluorescence polarization; fluorescence resonance energy transfer; kinase assay; kinase profiling; luminescence assays; radioisotope filtration binding assay; time-resolved fluorescence resonance energy transfer
22.  Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase 
Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their struture activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.
PMCID: PMC3139024  PMID: 21719286
Human peptide deformylase; Benzofuran-4,5-diones; Structure activity relationships; Fluorescence polarization; Antiproliferative agents
23.  Testing the Promiscuity of Commercial Kinase Inhibitors Against the AGC Kinase Group Using a Split-luciferase Screen 
Journal of Medicinal Chemistry  2012;55(4):1526-1537.
Using a newly developed competitive binding assay dependent upon the reassembly of a split reporter protein, we have tested the promiscuity of a panel of reported kinase inhibitors against the AGC group. Many non-AGC targeted kinase inhibitors target multiple members of the AGC group. In general, structurally similar inhibitors consistently exhibited activity toward the same target as well as toward closely related kinases. The inhibition data was analyzed to test the predictive value of either using identity scores derived from residues within 6 Å of the active site or identity scores derived from the entire kinase domain. The results suggest that the active site identity in certain cases may be a stronger predictor of inhibitor promiscuity. The overall results provide general guidelines for establishing inhibitor selectivity, as well as for the future design of inhibitors that either target or avoid AGC kinases.
PMCID: PMC3328412  PMID: 22257127
24.  ADAM17 cleaves CD16b (FcγRIIIb) in human neutrophils 
Biochimica et biophysica acta  2012;1833(3):680-685.
CD16b (FcγRIIIb) is exclusively expressed by human neutrophils and binds IgG in immune complexes. Cell surface CD16b undergoes efficient ectodomain shedding upon neutrophil activation and apoptosis. Indeed, soluble CD16b is present at high levels in the plasma of healthy individuals, which appears to be maintained by the daily turnover of apoptotic neutrophils. At this time, the principal protease responsible for CD16b shedding is not known. We show that CD16b plasma levels were significantly decreased in patients administered a selective inhibitor targeting the metalloproteases ADAM10 and ADAM17. Additional analysis with inhibitors selective for ADAM10 or ADAM17 revealed that only inhibition of ADAM17 significantly blocked the cleavage of CD16b following neutrophil activation and apoptosis. CD16b shedding by ADAM17 was further demonstrated using a unique ADAM17 function-blocking mAb and a cell-based ADAM17 reconstitution assay. Unlike human CD16, however, mouse CD16 did not undergo efficient ectodomain shedding upon neutrophil stimulation or apoptosis, indicating that this mechanism cannot be modeled in normal mice. Taken together, our findings are the first to directly demonstrate that ADAM17 cleaves CD16 in human leukocytes.
PMCID: PMC3556181  PMID: 23228566
Neutrophil; metalloprotease; ADAM17; apoptosis; Fc receptor
25.  Inhibition of Proliferation, Migration, and Matrix Metalloprotease Production in Malignant Mesothelioma Cells by Tyrosine Kinase Inhibitors1 
Neoplasia (New York, N.Y.)  2004;6(6):705-712.
The epidermal growth factor receptor (EGFR) is expressed in a variety of human solid tumors, including malignant mesothelioma. EGFR has been implicated in regulation of cell proliferation, survival, angiogenesis, and metastasis, making it an ideal target for drug development. ZD1839 (gefitinib) and OSI-774 (erlotinib) are new, low-molecular-weight, EGFR-selective tyrosine kinase (TK) inhibitors, whereas CI-1033 is a pan- EGFR family TK inhibitor. In the present study, we used ZD1839, OSI-774, and CI-1033 and investigated the effect of these drugs on proliferation, migration, and matrix metalloprotease (MMP) production in three malignant mesothelioma cell lines (M14K, ZL34, and SPC212). Using [3H]thymidine incorporation, DNA synthesis assay, we found that all three drugs inhibited transforming growth factor-alpha (TGF-α)-induced cellular proliferation in a dose-dependent manner. In addition, all three drugs induced apoptosis in ZL34 cells as determined by flow cytometry using annexin-V staining. Furthermore, all three drugs inhibited TGFA-α-induced cell migration (chemotaxis) in a dose-dependent manner as determined by Boyden chamber assay. TGF-α-induced MMP-9 production was also inhibited in a dose-dependent manner as determined by gelatin zymography in three cell lines tested. In conclusion, our study demonstrates inhibitory effectiveness of EGFR-TK inhibitors in malignant mesothelioma cells and suggests that these drugs may be an effective treatment strategy for malignant mesothelioma.
PMCID: PMC1531674  PMID: 15720796
Malignant mesothelioma; epidermal growth factor receptor (EGFR); tyrosine kinase (TK) inhibitors; migration; matrix metalloproteases (MMPs)

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