Protein flexibility and species specificity in structure-based drug discovery: dihydrofolate reductase as a test system

In structure-based drug discovery, researchers would like to identify all possible scaffolds for a given target. However, techniques that push the boundaries of chemical space could lead to many false positives or inhibitors that lack specificity for the target. Is it possible to broadly identify the appropriate chemical space for the inhibitors and yet maintain target specificity? To address this question, we have turned to dihydrofolate reductase (DHFR), a well-studied metabolic enzyme of pharmacological relevance. We have extended our multiple protein structure (MPS) method for receptor-based pharmacophore models to use multiple X-ray crystallographic structures. Models were created for DHFR from human and Pneumocystis carinii. These models incorporate a fair degree of protein flexibility and are highly selective for known DHFR inhibitors over drug-like non-inhibitors. Despite sharing a highly conserved active site, the pharmacophore models reflect subtle differences between the human and P. carinii forms, which identify species-specific, high-affinity inhibitors. We also use structures of DHFR from Candida albicans as a counter example. The available crystal structures show little flexibility, and the resulting models give poorer performance in identifying species-specific inhibitors. Therapeutic success for this system may depend on achieving species specificity between the related human host and these key fungal targets. The MPS technique is a promising advance for structure-based drug discovery for DHFR and other proteins of biomedical interest.     

A computational analysis of the binding model of MDM2 with inhibitors

Abstract  

It is a new and promising strategy for anticancer drug design to block the MDM2-p53 interaction using a non-peptide small-molecule inhibitor. We carry out molecular dynamics simulations to study the binding of a set of six non-peptide small-molecule inhibitors with the MDM2. The relative binding free energies calculated using molecular mechanics Poisson–Boltzmann surface area method produce a good correlation with experimentally determined results. The study shows that the van der Waals energies are the largest component of the binding free energy for each complex, which indicates that the affinities of these inhibitors for MDM2 are dominated by shape complementarity. The A-ligands and the B-ligands are the same except for the conformation of 2,2-dimethylbutane group. The quantum mechanics and the binding free energies calculation also show the B-ligands are the more possible conformation of ligands. Detailed binding free energies between inhibitors and individual protein residues are calculated to provide insights into the inhibitor-protein binding model through interpretation of the structural and energetic results from the simulations. The study shows that G1, G2 and G3 group mimic the Phe19, Trp23 and Leu26 residues in p53 and their interactions with MDM2, but the binding model of G4 group differs from the original design strategy to mimic Leu22 residue in p53.     

Library versus library recognition and inhibition of the HIV-1 Nef allelome

Rapid evolution of drug-resistant viruses renders essentially all small-molecule antiviral treatments ineffective. We demonstrate an in vitro library versus library approach to identify small molecules targeting a broad spectrum of HIV-1 Nef protein variants. The technique could provide more effective antiviral therapies. First, a library of clinically derived Nef allelic variants, termed an allelome, was selected for function by binding to Nef ligands p53, actin, or p56lck. Next, a library of small-molecule inhibitors challenged the Nef allelome in competition assays. In contrast to single-variant inhibition, structurally simpler molecules could better inhibit the Nef allelome. Additionally, Nef sequences selected for binding to p53 resembled sequences from patients with a rapid progression to AIDS phenotype. Thus, the allelome versus small-molecule library approach offers a route for improving antiviral drug discovery and elucidating fundamental mechanisms of viral pathogenesis and resistance.     

Prospective virtual screening for novel p53–MDM2 inhibitors using ultrafast shape recognition

The p53 protein, known as the guardian of genome, is mutated or deleted in approximately 50 % of human tumors. In the rest of the cancers, p53 is expressed in its wild-type form, but its function is inhibited by direct binding with the murine double minute 2 (MDM2) protein. Therefore, inhibition of the p53–MDM2 interaction, leading to the activation of tumor suppressor p53 protein presents a fundamentally novel therapeutic strategy against several types of cancers. The present study utilized ultrafast shape recognition (USR), a virtual screening technique based on ligand–receptor 3D shape complementarity, to screen DrugBank database for novel p53–MDM2 inhibitors. Specifically, using 3D shape of one of the most potent crystal ligands of MDM2, MI-63, as the query molecule, six compounds were identified as potential p53–MDM2 inhibitors. These six USR hits were then subjected to molecular modeling investigations through flexible receptor docking followed by comparative binding energy analysis. These studies suggested a potential role of the USR-selected molecules as p53–MDM2 inhibitors. This was further supported by experimental tests showing that the treatment of human colon tumor cells with the top USR hit, telmisartan, led to a dose-dependent cell growth inhibition in a p53-dependent manner. It is noteworthy that telmisartan has a long history of safe human use as an approved anti-hypertension drug and thus may present an immediate clinical potential as a cancer therapeutic. Furthermore, it could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53–MDM2 inhibitors against variety of cancers. Importantly, the present study demonstrates that the adopted USR-based virtual screening protocol is a useful tool for hit identification in the domain of small molecule p53–MDM2 inhibitors.     

Structures of Clostridium botulinum Neurotoxin Serotype A Light Chain complexed with small-molecule inhibitors highlight active-site flexibility

The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.     

Activity-Directed Synthesis of Inhibitors of the p53/hDM2 Protein–Protein Interaction

Protein–protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural-diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity-directed synthesis (ADS), a function-driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small-molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors.     

Globally Approved EGFR Inhibitors: Insights into Their Syntheses,Target Kinases,Biological Activities,Receptor Interactions,and Metabolism

Targeting the EGFR with small-molecule inhibitors is a confirmed valid strategy in cancer therapy. Since the FDA approval of the first EGFR-TKI, erlotinib, great efforts have been devoted to the discovery of new potent inhibitors. Until now, fourteen EGFR small-molecule inhibitors have been globally approved for the treatment of different types of cancers. Although these drugs showed high efficacy in cancer therapy, EGFR mutations have emerged as a big challenge for these drugs. In this review, we focus on the EGFR small-molecule inhibitors that have been approved for clinical uses in cancer therapy. These drugs are classified based on their chemical structures, target kinases, and pharmacological uses. The synthetic routes of these drugs are also discussed. The crystal structures of these drugs with their target kinases are also summarized and their bonding modes and interactions are visualized. Based on their binding interactions with the EGFR, these drugs are also classified into reversible and irreversible inhibitors. The cytotoxicity of these drugs against different types of cancer cell lines is also summarized. In addition, the proposed metabolic pathways and metabolites of the fourteen drugs are discussed, with a primary focus on the active and reactive metabolites. Taken together, this review highlights the syntheses, target kinases, crystal structures, binding interactions, cytotoxicity, and metabolism of the fourteen globally approved EGFR inhibitors. These data should greatly help in the design of new EGFR inhibitors.     

Structure-based design of potent non-peptide MDM2 inhibitors

A successful structure-based design of a class of non-peptide small-molecule MDM2 inhibitors targeting the p53-MDM2 protein-protein interaction is reported. The most potent compound 1d binds to MDM2 protein with a Ki value of 86 nM and is 18 times more potent than a natural p53 peptide (residues 16-27). Compound 1d is potent in inhibition of cell growth in LNCaP prostate cancer cells with wild-type p53 and shows only a weak activity in PC-3 prostate cancer cells with a deleted p53. Importantly, 1d has a minimal toxicity to normal prostate epithelial cells. Our studies provide a convincing example that structure-based strategy can be employed to design highly potent, non-peptide, cell-permeable, small-molecule inhibitors to target protein-protein interaction, which remains a very challenging area in chemical biology and drug design.     

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.     

Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides

Peptide stapling is a method for designing macrocyclic alpha‐helical inhibitors of protein–protein interactions. However, obtaining a cell‐active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain‐promoted azide–alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell‐active stapled peptides. As a proof of concept, MDM2‐binding peptides were stapled in parallel, directly in cell culture medium in 96‐well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α‐Helicity was confirmed by a crystal structure of the MDM2‐peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high‐throughput biological applications.     

Development of a potent Bcl-x(L) antagonist based on alpha-helix mimicry

The rational design of low-molecular weight ligands that disrupt protein-protein interactions is still a challenging goal in medicinal chemistry. Our approach to this problem involves the design of molecular scaffolds that mimic the surface functionality projected along one face of an alpha-helix. Using a terphenyl scaffold, which in a staggered conformation closely reproduces the projection of functionality on the surface of an alpha-helix, we designed mimics of the pro-apoptotic alpha-helical Bak-peptide as inhibitors of the Bak/Bcl-xL interaction. This led to the development of a potent Bcl-xL antagonist (KD = 114 nM), whose binding affinity for Bcl-xL was assessed by a fluorescence polarization assay. To determine the binding site of the developed inhibitor we used docking studies and an HSQC-NMR experiment with 15N-labeled Bcl-xL protein. These studies suggest that the inhibitor is binding in the same hydrophobic cleft as the Bak- and Bad-peptides.     

Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase.

BACKGROUND: Group I beta-lactamases are a major cause of antibiotic resistance to beta-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. RESULTS: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K(i) 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K(i) values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 A resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. CONCLUSIONS: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta-lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K(i) 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta-lactamase.     

CONCEPTS: New dynamic algorithm for de novo drug suggestion

We describe a new method for de novo design of molecules that bind to protein active sites. The method, CONCEPTS (Creation of Novel Compounds by Evaluation of Particles at Target Sites), places a group of atom-like particles in the site. The particles are free to move within the site to improve binding to the protein. A key innovation of this technique is that covalent connections are made among the particles in a stochastic and dynamically reversible manner. These changes in the topology are either accepted or rejected depending on their ability to improve the total energy of the enzyme–inhibitor complex. The method is applied to two test systems: The FK506 binding protein (FKBP-12) and HIV-1 aspartyl protease. In both cases, we are able to predict, de novo, drugs that have striking similarities to known potent inhibitors and that can successfully be used to generate “hits” of the known inhibitors from a data base. © John Wiley & Sons, Inc.     

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.     

Self-assembling small molecules form nanofibrils that bind procaspase-3 to promote activation

Modulating enzyme function with small-molecule activators, as opposed to inhibitors, offers new opportunities for drug discovery and allosteric regulation. We previously identified a compound, called 1541, from a high-throughput screen (HTS) that stimulates activation of a proenzyme, procaspase-3, to generate mature caspase-3. Here we further investigate the mechanism of activation and report the surprising finding that 1541 self-assembles into nanofibrils exceeding 1 μm in length. These particles are an unanticipated outcome from an HTS that have properties distinct from standard globular protein aggregators. Moreover, 1541 nanofibrils function as a unique biocatalytic material that activates procaspase-3 via induced proximity. These studies demonstrate a novel approach for proenzyme activation through binding to fibrils, which may mimic how procaspases are naturally processed on protein scaffolds.     

Structure-based grafting and identification of kinase–inhibitors to target mTOR signaling pathway as potential therapeutics for glioblastoma

Mammalian target of rapamycin (mTOR), a key mediator of PI3K/Akt/mTOR signaling pathway, has recently emerged as a compelling molecular target in glioblastoma. The mTOR is a member of serine/threonine protein kinase family that functions as a central controller of growth, proliferation, metabolism and angiogenesis, but its signaling is dysregulated in various human diseases especially in certain solid tumors including the glioblastoma. Here, considering that there are various kinase inhibitors being approved or under clinical or preclinical development, it is expected that some of them can be re-exploited as new potent agents to target mTOR for glioblastoma therapy. To achieve this, a synthetic pipeline that integrated molecular grafting, consensus scoring, virtual screening, kinase assay and structure analysis was described to systematically profile the binding potency of various small-molecule inhibitors deposited in the protein kinase–inhibitor database against the kinase domain of mTOR. Consequently, a number of structurally diverse compounds were successfully identified to exhibit satisfactory inhibition profile against mTOR with IC₅₀ values at nanomolar level. In particular, few sophisticated kinase–inhibitors as well as a flavonoid myricetin showed high inhibitory activities, which could thus be considered as potential lead compounds to develop new potent, selective mTOR–inhibitors. Structural examination revealed diverse nonbonded interactions such as hydrogen bonds, hydrophobic forces and van der Waals contacts across the complex interface of mTOR with myricetin, conferring both stability and specificity for the mTOR–inhibitor binding.     

Virtual screening studies to design potent CDK2-cyclin A inhibitors

The cell division cycle is controlled by cyclin-dependent kinases (CDK), which consist of a catalytic subunit (CDK1-CDK8) and a regulatory subunit (cyclin A-H). Pharmacophore analysis indicates that the best inhibitor model consists of (1) two hydrogen bond acceptors, (2) one hydrogen bond donor, and (3) one hydrophobic feature. The HypoRefine pharmacophore model gave an enrichment factor of 1.31 and goodness of fit score of 0.76. Docking studies were carried out to explore the structural requirements for the CDK2-cyclin A inhibitors and to construct highly predictive models for the design of new inhibitors. Docking studies demonstrate the important role of hydrogen bond and hydrophobic interactions in determining the inhibitor-receptor binding affinity. The validated pharmacophore model is further used for retrieving the most active hits/lead from a virtual library of molecules. Subsequently, docking studies were performed on the hits, and novel series of potent leads were suggested based on the interaction energy between CDK2-cyclin A and the putative inhibitors.     

Computational discovery of picomolar Q(o) site inhibitors of cytochrome bc1 complex

A critical challenge to the fragment-based drug discovery (FBDD) is its low-throughput nature due to the necessity of biophysical method-based fragment screening. Herein, a method of pharmacophore-linked fragment virtual screening (PFVS) was successfully developed. Its application yielded the first picomolar-range Q(o) site inhibitors of the cytochrome bc(1) complex, an important membrane protein for drug and fungicide discovery. Compared with the original hit compound 4 (K(i) = 881.80 nM, porcine bc(1)), the most potent compound 4f displayed 20?507-fold improved binding affinity (K(i) = 43.00 pM). Compound 4f was proved to be a noncompetitive inhibitor with respect to the substrate cytochrome c, but a competitive inhibitor with respect to the substrate ubiquinol. Additionally, we determined the crystal structure of compound 4e (K(i) = 83.00 pM) bound to the chicken bc(1) at 2.70 ? resolution, providing a molecular basis for understanding its ultrapotency. To our knowledge, this study is the first application of the FBDD method in the discovery of picomolar inhibitors of a membrane protein. This work demonstrates that the novel PFVS approach is a high-throughput drug discovery method, independent of biophysical screening techniques.