Human African trypanosomiasis (HAT) is a major health problem in sub-Saharan Africa caused by Trypanosoma brucei infection. Current HAT drugs are difficult to administer and not effective against all parasite species at different stages of the disease which indicates an unmet pharmaceutical need. TbRET2 is an indispensable enzyme for the parasite, and is targeted here using a computational approach that combines molecular dynamics simulations and virtual screening. The compounds prioritized are then tested in T. brucei via Alamar blue cell viability assays. This work identified 20 drug-like compounds which are candidates for further testing in the drug discovery process.
TUTase; virtual screening; RET2; trypanosomes; Trypanosoma brucei; human African trypanosomiasis; sleeping sickness; HAT
Pharmacologic reinduction of the developmentally silenced fetal (γ) globin genes has been achieved in hemoglobinopathy patients using short chain fatty acid derivatives, with therapeutic effects. However, higher-potency inducers than are available in currently identified short chain fatty acid derivatives are desirable for long-term use. Using several short-chain fatty acids with established γ-globin induction activity, a pharmacophore template was constructed with the TFIT module of the flo software and used to select several new candidate compounds, three of which exhibited significant activity in a γ-globin gene reporter transcriptional assay which detects only strong inducers. The data were used to construct a new pharmacophore and a ‘pseudo’ receptor around it. Six hundred and thirty low-molecular weight compounds were docked into this receptor model. Of 26 compounds selected and tested in functional assays, two compounds showed activity >500% over control levels and two had activity 200% over control range, significantly more active than previously identified short chain fatty acid derivative fetal globin gene inducers. Three compounds had less activity; the remainder showed moderate activity. These findings demonstrate the feasibility of using iterative construction of pharmacophores, pseudo-binding site modeling, and virtual screening to identify small molecules with the ability to induce transcription of specific target genes, for potential therapeutics.
pharmacophore; pseudo receptor; fetal globin; FLO/QXP
Using small, flat aromatic rings as components of fragments or molecules is a common practice in fragment-based drug discovery and lead optimization. With an increasing focus on the exploration of novel biological and chemical space, and their improved synthetic accessibility, 3D fragments are attracting increasing interest. This study presents a detailed analysis of 3D and 2D ring fragments in marketed drugs. Several measures of properties were used, such as the type of ring assemblies and molecular shapes. The study also took into account the relationship between protein classes targeted by each ring fragment, providing target-specific information. The analysis shows the high structural and shape diversity of 3D ring systems and their importance in bioactive compounds. Major differences in 2D and 3D fragments are apparent in ligands that bind to the major drug targets such as GPCRs, ion channels, and enzymes.
drug; fragment; lead optimization; ring
The AKT and NF-κB pathways are central regulators of cellular signaling events at the basis of tumor development and progression. Both pathways are often up-regulated in different tumor types including melanoma. We recently reported the identification of compound 1 (BI-69A11) as inhibitor of the AKT and the NF-κB pathways. Here we describe SAR studies that led to novel fluorinated derivatives with increased cellular potency, reflected in efficient inhibition of AKT and IKKs. Selected compounds demonstrated effective toxicity on melanoma, breast and prostate cell lines. Finally, a representative derivative showed promising efficacy in an in vivo melanoma xenograft model.
Staphylococcus aureus is the leading cause of hospital-acquired infections in the United States. The emergence of multi-drug resistant strains of S. aureus has created an urgent need for new antibiotics. S. aureus uses the sortase A (SrtA) enzyme to display surface virulence factors suggesting that compounds that inhibit its activity will function as potent anti-infective agents. Here we report the identification of several inhibitors of SrtA using virtual screening methods that employ the relaxed complex scheme, an advanced computer-docking methodology that accounts for protein receptor flexibility. Experimental testing validates that several compounds identified in the screen inhibit the activity of SrtA. A lead compound based on the 2-phenyl-2,3-dihydro-1H-perimidine scaffold is particularly promising and its binding mechanism was further investigated using molecular dynamics simulations and by conducting preliminary structure activity relationship studies.
Staphylococcus aureus; MRSA; sortase; SrtA; transpeptidation; Gram-positive; drug discovery; virtual screening; relaxed complex scheme; molecular dynamics; docking
In our previous study we developed a novel series of tetrahydroisoquinoline-based hydroxamic acid derivatives as histone deacetylase (HDAC) inhibitors (Bioorg. Med. Chem., 2010, 18, 1761–1772., J. Med. Chem., 2011, 54, 2823–2838.), among which compound ZYJ-34c (1) was identified and validated as the most potent one with marked in vitro and in vivo antitumor potency (J. Med. Chem., 2011, 54, 5532–5539.). Herein further modification of 1 afforded another oral active analogue ZYJ-34v (2) with simplified structure and lower molecular weight. Biological evaluation of compound 2 showed efficacious inhibition against HDAC1, 2, 3 and 6, which was confirmed by western blot analysis results. Most importantly, compound 2 exhibited similar even more potent in vitro and in vivo antitumor activities relative to the approved HDAC inhibitor SAHA.
Histone deacetylases; Inhibitor; Tetrahydroisoquinoline; Valproic acid; Oral active; Antitumor
The cancer stem cell marker, EpCAM, is an important indicator of wnt-β-catenin signaling activation and a functional component of hepatocellular tumor initiating cells. A high-throughput screening assay was developed to identify inhibitors of EpCAM-dependent growth of hepatocellular carcinoma cells. EpCAM(+) and EpCAM(−) HCC cell lines were assessed for differential sensitivity to a wnt-β-catenin pathway inhibitor. Libraries comprising 22,668 pure compounds and 107,741 crude or partially purified natural product extracts were tested and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express EpCAM), but resulted in generation of cell populations expressing lower levels of EpCAM. Sublethal concentrations (~IC50) reduced median EpCAM expression to 28% of control after 1 d and 19% of control after 2 d. Reduction in EpCAM expression preceded growth inhibition suggesting that a threshold of EpCAM expression may be required for growth of EpCAM-dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM-dependent cell growth.
Hepatocellular carcinoma (HCC); EpCAM (epithelial cell adhesion molecule); wnt/β-catenin; high-throughput screening (HTS)
Targeting co-stimulatory molecules to modulate the immune response has
been shown to have useful therapeutic effects for autoimmune diseases. Among the
co-stimulatory molecules, CD2 and CD58 are very important in the early stages of
generation of an immune response. Our goal was to utilize CD2-derived peptides
to modulate protein-protein interactions between CD2 and CD58, thereby
modulating the immune response. Several peptides were designed based on the
structure of the CD58 binding domain of CD2 protein. Among the CD2-derived
peptides, peptide 6 from the F and C β-strand region of CD2 protein
exhibited inhibition of cell-cell adhesion in the nanomolar concentration range.
Peptide 6 was evaluated for its ability to bind to CD58 in Caco-2 cells and to
CD48 in T cells from rodents. A molecular model was proposed for binding a
peptide to CD58 and CD48 using docking studies. Furthermore, in
vivo studies were carried out to evaluate the therapeutic ability
of the peptide to modulate the immune response in the collagen-induced arthritis
(CIA) mouse model. In vivo studies indicated that peptide 6 was
able to suppress the progression of CIA. Evaluation of the antigenicity of
peptides in CIA and transgenic animal models indicated that this peptide is not
CD2; CD58; collagen-induced arthritis; cyclic peptide; docking; immune response; protein-protein interaction
Twelve alkyl analogues (1 - 12) of the high-affinity serotonin transporter (SERT) inhibitor 6-nitroquipazine (6-NQ) were synthesised and studied using in vitro radioligand competition binding assays to determine their binding affinity (Ki). The putative antidepressant activity of five of the binders with the highest SERT binding affinities was studied by the forced swim and locomotor activity mouse tests. The three dimensional (3D) structures of 8 and 9 were determined using NOE NMR technique. Flexible docking of the compounds was undertaken to illustrate the binding of the compounds in the SERT model. Our results showed that several of the 6-NQ analogues are high-affinity SERT inhibitors and indicated that the octyl (8), decyl (10) and dodecyl (12) 6-NQ analogues exhibit moderate antidepressant activity.
Serotonin transporter; 6-nitroquipazine alkyl analogues; Radioligand competition assay; NMR spectroscopy; Flexible docking; Porsolt forced swim test
Mithramycin; Mithramycin SA; Semi-synthesis; Anti-cancer; Aureolic acid
African trypanosomiasis is a neglected tropical disease affecting humans and animals across 36 sub-Saharan African countries. We have investigated the potential to exploit a ‘piggyback’ approach to inhibit Trypanosoma brucei transmission by targeting the key developmental regulator of transmission, T. brucei protein tyrosine phosphatase 1. This strategy took advantage of the extensive investment in inhibitors for human protein tyrosine phosphatase 1B, a key target for pharmaceutical companies for the treatment of obesity and diabetes. Structural predictions for human and trypanosome tyrosine phosphatases revealed the overall conservation of important functional motifs, validating the potential for exploiting cross specific compounds. Thereafter, nineteen inhibitors were evaluated; seventeen from a protein tyrosine phosphatase 1B-targeted inhibitor library and two from literature analysis – oleanolic acid and suramin, the latter of which is a front line drug against African trypanosomiasis. The compounds tested displayed similar inhibitory activities against the human and trypanosome enzymes, mostly behaving as noncompetitive inhibitors. However, their activity against T. brucei in culture was low, necessitating further chemical modification to improve their efficacy and specificity. Nonetheless, the results validate the potential to explore a ‘piggyback’ strategy targeting T. brucei protein tyrosine phosphatase 1 through exploiting the large pharmacological investment in therapies for obesity targeting protein tyrosine phosphatase 1B.
biological screening; drug discovery; kinase; phosphatase
Fifty analogues of batzelladine K were synthesized and evaluated for in vitro antimalarial (Plasmodium falciparum), antileishmanial (Leishmania donovani), antimicrobial (panel of bacteria and fungi), antiviral (HIV-1) activities. Analogues 14h and 20l exhibited potential antimalarial activity against chloroquine-sensitive D6 strain with IC50 1.25 and 0.88 μM and chloroquine-resistant W2 strain with IC50 1.64 and 1.07 μM, respectively. Analogues 12c and 14c having nonyl substitution showed the most potent antileishmanial activity with IC50 2.39 and 2.78 μM and IC90 11.27 and 12.76 μM respectively. Three analogues 12c, 14c and 14i were the most active against various pathogenic bacteria and fungi with IC50 <3.02 μM and MIC/MBC/MFC <6 μM. Analogue 20l having pentyl and methyl substituents on tricycle showed promising activities against all pathogens. However, none was found active against HIV-1. Our study demonstrated that the tricyclic guanidine compounds provide new structral class for broad spectrum activity.
Batzelladine; tricyclic guanidine; antimalarial; antileishmanial; antimicrobial; anti-HIV
α/β-hydrolase domain-containing 6 (ABHD6) represents a potentially attractive therapeutic target for indirectly potentiating 2-arachidonoylglycerol signaling, however, the enzyme is currently largely uncharacterized. Here we describe a five element, ligand-based pharmacophore model along with a refined homology model of ABHD6. Following a virtual screen of a modest database, both the pharmacophore and homology models were found to be highly predictive, preferentially identifying ABHD6 inhibitors over druglike noninhibitors. The models yield insight into the features required for optimal ligand binding to ABHD6 and the atomic structure of the binding site. In combination the two models should be very helpful not only in high throughput virtual screening, but also in lead optimization, and will facilitate the development of novel, selective ABHD6 inhibitors as potential drugs.
We have developed competitive and direct binding methods to examine small-molecule inhibitors of protein tyrosine phosphatase (PTPase) activity. Focusing on the Yersinia outer protein H (YopH), a potent bacterial PTPase, we describe how an understanding of the kinetic interactions involving YopH, peptide substrates, and small-molecule inhibitors of PTPase activity can be beneficial for inhibitor screening and we further translate these results into a microarray assay for high-throughput screening.
Affinity screening; competitive binding assay; high-throughput screening; protein tyrosine phosphatase; peptide microarray; phosphopeptide; surface plasmon resonance (SPR); Yersinia pestis outer protein H (YopH)
Amyloid binding alcohol dehydrogenase (ABAD), a mitochondrial protein, is a cofactor facilitating amyloid-β peptide (Aβ) induced cell stress. Antagonizing Aβ-ABAD interaction protects against aberrant mitochondrial and neuronal function and improves learning memory in the Alzheimer’s disease (AD) mouse model. Therefore, it offers a potential target for Alzheimer’s drug design, by identifying potential inhibitors of Aβ-ABAD interaction. 2D QSAR methods were applied to novel compounds with known IC50 values, which formed a training set. A correlation analysis was carried out comparing the statistics of the measured IC50 with predicted values. These selectivity-determining descriptors were interpreted graphically in terms of principle component analyses, which are highly informative for the lead optimization process with respect to activity enhancement. A 3D pharmacophore model also was created. The 2D QSAR and 3D pharmacophore models will assist in hi-throughput screening. In addition, ADME descriptors were also determined to study their pharmacokinetic properties. Finally, ABAD molecular docking study of these novel molecules was undertaken to determine whether these compounds exhibit significant binding affinity with the binding site. We have synthesized only the compounds that have shown the best drug like properties as candidates for further studies.
Molecular Docking; Quantitative Structure Activity Relationship; ADME Prediction; ABAD Inhibitors; Benzothiazole Aminophosphonates
Since protein/protein interactions usually trigger signaling processes, inhibitors of those interactions must preclude protein binding without eliciting the signaling process themselves. To accomplish those goals, small molecules need to target those protein residues that contribute the most to binding (binding hotspots) without disturbing those residues that initiate signaling processes (allosteric hotspots). The availability of a blueprint identifying binding and allosteric hotspots will significantly aid inhibitor design and optimization. In this paper, we show that in some situations the blueprint can be constructed by combining the standard technique of alanine scanning mutagenesis with isothermal titration calorimetry (ITC). We demonstrate the approach by developing the combined binding and allosteric hotspots blueprint for CD4/gp120, the initial interaction leading to HIV-1 cell infection. A major finding of these studies is that not all binding hotspots are allosteric hotspots opening the possibility for the rational design of inhibitors and antagonist or agonist modulators.
Binding Affinity; Enthalpy; Entropy; Thermodynamic Optimization; Isothermal Titration Calorimetry; Alanine Scanning Mutagenesis
It is widely recognized that ADMET (Adsorption, Distribution, Metabolism, Excretion - Toxicology) liabilities kill the majority of drug candidates that progress to clinical trials. The development of computational models to predict small molecule membrane permeability is therefore of considerable scientific and public health interest. Empirical qualitative structure permeability relationship (QSPR) models of permeability have been a mainstay in industrial applications, but lack a deep understanding of the underlying biological physics. Others and we have shown that implicit solvent models to predict passive permeability for small molecules exhibit mediocre predictive performance when validated across experimental test sets. Given the vast increase in computer power, more efficient parallelization schemes, and extension of current atomistic simulation codes to general use graphical processing units (GPUs), the development and application of physical models based on all-atom simulations may now be feasible. Preliminary results from rigorous free energy calculations using all-atom simulations indicate that performance relative to implicit solvent models may be improved, but many outstanding questions remain. Here we review the current state of the art physical models for passive membrane permeability prediction, and present a prospective look at promising new directions for all-atom approaches.
Passive membrane permeability; Implicit solvent; Molecular Dynamics; Homogenous solubility model
Following sequencing and assembly of the human genome, the preferred methods for identification of new drug targets have changed dramatically. Modern tactics such as genome-wide association studies (GWAS) and deep sequencing are fundamentally different from the pharmacology-guided approaches used previously, in which knowledge of small molecule ligands acting at their cellular targets was the primary discovery engine. A consequence of the “target-first, pharmacology-second” strategy is that many predicted drug targets are non-enzymes, such as scaffolding, regulatory or structural proteins, and their activities are often dependent on protein-protein interactions (PPIs). These types of targets create unique challenges to drug discovery efforts because enzymatic turnover cannot be used as a convenient surrogate for compound potency. Moreover, it is often challenging to predict how ligand binding to non-enzymes might affect changes in protein function and/or pathobiology. Thus, in the post-genomic era, targets might be strongly implicated by molecular biology-based methods, yet they often later earn the designation of “undruggable.” Can the scope of available targets be widened to include these promising, but challenging, non-enzymes? In this review, we discuss advances in high throughput screening technology and chemical library design that are emerging to deal with these challenges.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcription factor of the antioxidant response element (ARE) pathway, coordinating the induction of detoxifying and antioxidant enzymes. Nrf2 is normally sequestered in the cytoplasm by Kelch-like ECH associating protein 1 (Keap1). To identify novel small molecules that will disturb Nrf2:Keap1 binding and promote activation of the Nrf2-ARE pathway, we generated a quantum model based on the structures of known Nrf2-ARE activators. We used the quantum model to perform in silico screening on over 18 million commercially available chemicals to identify the structures predicted to activate the Nrf2-ARE pathway based on the quantum model. The top hits were tested in vitro and half of the predicted hits activated the Nrf2-ARE pathway significantly in primary cell culture. In addition, we identified a new family of Nrf2-ARE activating structures that all have comparable activity to tBHQ and protect against oxidative stress and dopaminergic toxins in vitro. The improved ability to identify potent activators of Nrf2 through the combination of in silico and in vitro screening described here improves the speed and cost associated with screening Nrf2-ARE activating compounds for drug development.
The relaxed complex scheme is an in silico drug
screening method that accounts for receptor flexibility by using molecular
dynamics simulations. Here, we used this approach combined with similarity
searches and experimental inhibition assays to identify several low micro-molar,
non-bisphosphonate inhibitors, bisamidines, of farnesyl diphosphate synthase
(FPPS), an enzyme targeted by some anti-cancer and anti-microbial agents and for
the treatment of bone resorption diseases. This novel class of FPPS inhibitors
have more drug-like properties than existing bisphosphonate inhibitors, making
them interesting pharmaceutical leads.
HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical-shift changes of RNH with those of RNH dimer, in the presence and absence of Mg2+, were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the “substrate-handle region”). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong, el (2011) Chem. Biol. Drug Des. 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.
Reverse transcriptase; ribonuclease H; HIV; acylhydrazone; inhibitor; enzyme; protein; NMR; dimer; molecular docking
H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA), is a synthetic tetrapeptide with extraordinary selectivity for the mu-opioid receptor and is an extremely potent analgesic. [Dmt1]DALDA is unusual in the way that the greater part of its analgesic potency appears to be produced by its actions in the spinal cord. Furthermore, [Dmt1]DALDA inhibits norepinephrine re-uptake and is a mitochondria-targeted antioxidant. Such characteristics may make [Dmt1]DALDA particularly effective against neuropathic pain. The present study was designed to compare the effects of [Dmt1]DALDA and morphine on thermal hyperalgesia in an experimental neuropathic pain model. Neuropathic pain was induced in rats by surgical ligation of the L5 spinal nerve, and thermal pain thresholds were assessed by latencies of paw withdrawal to radiant heat. The increase in paw withdrawal latency was greater after the administration of [Dmt1]DALDA than that of morphine in neuropathic rats at doses that were equianalgesic in naïve animals. We conclude that [Dmt1]DALDA is more effective than morphine against thermal hyperalgesia in this experimental model of neuropathic pain.
Opioid; neuropathic pain; hyperalgesia; [Dmt1]DALDA; morphine
Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored affinity and efficacy profiles. In particular, we have obtained pentapeptides 8, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]NH2, and 12, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]OH, which demonstrates high affinity and full agonist behavior at MOR, high affinity but very low efficacy for DOR, and minimal affinity for the kappa opioid receptor (KOR). Functional properties of these peptides as MOR agonists/DOR antagonists lacking undesired KOR activity make them promising candidates for future in vivo studies of MOR/DOR interactions. Subtle structural variation of 12, by substituting D-Cys5 for L-Cys5, generated analog 13 which maintains low nanomolar MOR and DOR affinity, but which displays no efficacy at either receptor. These results demonstrate the power and utility of accurate receptor models for structure-based ligand design, as well as the profound sensitivity of ligand function on its structure.
Delta opioid receptor; G protein-coupled receptors; mixed efficacy ligand; mu opioid receptor; opioid; peptide; structure-based design