Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 µM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.

Role of antibiotic ligand in nascent peptide-dependent ribosome stalling.

Specific nascent peptides in the ribosome exit tunnel can elicit translation arrest. Such ribosome stalling is used for regulation of expression of some bacterial and eukaryotic genes. The stalling is sensitive to additional cellular cues, most commonly the binding of specific small-molecular-weight cofactors to the ribosome. The role of cofactors in programmed translation arrest is unknown. By analyzing nascent peptide- and antibiotic-dependent ribosome stalling that controls inducible expression of antibiotic resistance genes in bacteria, we have found that the antibiotic is directly recognized as a part of the translation modulating signal. Even minute structural alterations preclude it from assisting in ribosome stalling, indicating the importance of precise molecular interactions of the drug with the ribosome. One of the sensors that monitor the structure of the antibiotic is the 23S rRNA residue C2610, whose mutation reduces the efficiency of nascent peptide- and antibiotic-dependent ribosome stalling. These findings establish a new paradigm of the role of the cofactor in programmed translation arrest in which a small molecule is recognized along with specific nascent peptide sequences as a composite structure that provokes arrest of translation. A similar mechanism could be used by the ribosome to sense a variety of cellular metabolites.

Structure of dihydroorotase from Bacillus anthracis at 2.6†Šresolution.

Dihydroorotase (EC 3.5.2.3) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate to L-dihydroorotate in the third step of the pyrimidine-biosynthesis pathway in Bacillus anthracis. A comparison is made between the structures of dihydroorotase from four different organisms, including B. anthracis dihydroorotase, and reveals substantial variations in the active site, dimer interface and overall tertiary structure. These differences demonstrate the utility of exploring multiple structures of a molecular target as expressed from different organisms and how these differences can be exploited for structure-based drug discovery.

Structural model of a complex between the heterotrimeric G protein, Gsalpha, and tubulin.

A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase.

Inhibition of MptpB phosphatase from Mycobacterium tuberculosis impairs mycobacterial survival in macrophages.

OBJECTIVES: The secreted Mycobacterium tuberculosis protein tyrosine phosphatase (MptpB) is a virulence factor for M. tuberculosis and contributes to its survival within host macrophages. The aim of this study was to identify potent selective inhibitors of MptpB and to determine the efficacy of these compounds in mycobacterium-infected macrophages. METHODS: The inhibitory effect of a small library of compounds on MptpB was first examined in vitro. The efficacy of these compounds was further examined in mycobacterium-infected macrophages. RESULTS: We have identified a new family of double-site isoxazole-based compounds that are potent selective inhibitors of MptpB. Importantly, the inhibitors substantially reduce mycobacterial survival in infected macrophages. In contrast with current anti-tubercular drugs, these MptpB inhibitors do not have bactericidal action but rather, severely impair mycobacterial growth within macrophages. Docking analysis suggests a double-site binding mechanism of inhibition with the isoxazole head in the active site and a salicylate group in a secondary binding pocket that is a unique structural feature of MptpB. CONCLUSIONS: These results provide the first evidence that inhibition of phosphatases can be exploited against mycobacterial infections. The cell activity of the inhibitors together with the lack of MptpB human orthologues suggests a strong potential for these compounds to be developed as drug candidates against tuberculosis and promises a new therapeutic strategy to tackle clearance and reduce the persistence of M. tuberculosis infection.

Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design.

Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 A and 2.0 A. Both enzymes are dimers with monomers arranged in a "tail-to-tail" orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases.

Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors.

Design, synthesis and biological evaluation of a series of 5-chloropyridine ester-derived severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of the carboxylate functionality is critical to potency. Inhibitor 10 with a 5-chloropyridinyl ester at position 4 of the indole ring is the most potent inhibitor with a SARS-CoV 3CLpro IC(50) value of 30 nM and an antiviral EC(50) value of 6.9 microM. Molecular docking studies have provided possible binding modes of these inhibitors.

Design and synthesis of 2-pyridones as novel inhibitors of the Bacillus anthracis enoyl-ACP reductase.

Enoyl-ACP reductase (ENR), the product of the FabI gene, from Bacillus anthracis (BaENR) is responsible for catalyzing the final step of bacterial fatty acid biosynthesis. A number of novel 2-pyridone derivatives were synthesized and shown to be potent inhibitors of BaENR.

Regioselective covalent modification of hemoglobin in search of antisickling agents.

Although the molecular defect in sickle hemoglobin that produces sickle cell disease has been known for decades, there is still no effective drug treatment that acts on hemoglobin itself. In this work, a series of diversely substituted isothiocyanates (R-NCS) were examined for their regioselective reaction with hemoglobin in an attempt to alter the solubility properties of sickle hemoglobin. Electrospray mass spectrometry, molecular modeling, X-ray crystallography, and conventional protein chemistry were used to study this regioselectivity and the resulting increase in solubility of the modified hemoglobin. Depending on the attached R-group, the isothiocyanates were found to react either with the Cysbeta93 or the N-terminal amine of the alpha-chain. One of the most effective compounds in the series, 2-(N,N-dimethylamino)ethyl isothiocyanate, selectively reacts with the thiol of Cysbeta93 which, in conjunction with the cationic group, was seen to perturb the local hemoglobin structure. This modified HbS shows an approximately 30% increase in solubility for the fully deoxygenated state, along with a significant increase in oxygen affinity. This compound and a related analogue appear to readily traverse the erythrocyte membrane. A discussion of the relation of these structural changes to inhibition of gelation is presented. The dual activities of increasing HbS oxygen affinity and directly inhibiting deoxy HbS polymerization, in conjunction with facile membrane traversal, suggest that these cationic isothiocyanates show substantial promise as lead compounds for development of therapeutic agents for sickle cell disease.

Severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand X-ray structure and biological evaluation.

The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead optimization and structure-activity studies to provide a series of improved inhibitors that show potent PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the (S)-Me inhibitor 15 h (enzyme IC(50) = 0.56 microM; antiviral EC(50) = 9.1 microM) and the corresponding (R)-Me 15 g (IC(50) = 0.32 microM; antiviral EC(50) = 9.1 microM) are the most potent compounds in this series, with nearly equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15 g-bound SARS-CoV PLpro and a corresponding model of 15 h docked to PLpro provide intriguing molecular insight into the ligand-binding site interactions.

Structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome-coronavirus papain-like protease.

We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, nonpeptide inhibitors of SARS-CoV PLpro. Our initial lead compound was identified via high-throughput screening of a diverse chemical library. We subsequently carried out structure-activity relationship studies and optimized the lead structure to potent inhibitors that have shown antiviral activity against SARS-CoV infected Vero E6 cells. Upon the basis of the X-ray crystal structure of inhibitor 24-bound to SARS-CoV PLpro, a drug design template was created. Our structure-based modification led to the design of a more potent inhibitor, 2 (enzyme IC(50) = 0.46 microM; antiviral EC(50) = 6 microM). Interestingly, its methylamine derivative, 49, displayed good enzyme inhibitory potency (IC(50) = 1.3 microM) and the most potent SARS antiviral activity (EC(50) = 5.2 microM) in the series. We have carried out computational docking studies and generated a predictive 3D-QSAR model for SARS-CoV PLpro inhibitors.

Design and synthesis of aryl ether inhibitors of the Bacillus anthracis enoyl-ACP reductase.

The problem of increasing bacterial resistance to the current generation of antibiotics is well documented. Known resistant pathogens such as methicillin-resistant Staphylococcus aureus are becoming more prevalent, while the potential exists for developing drug-resistant pathogens for use as bioweapons, such as Bacillus anthracis. The biphenyl ether antibacterial agent, triclosan, exhibits broad-spectrum activity by targeting the fatty acid biosynthetic pathway through inhibition of enoyl-acyl carrier protein reductase (ENR) and provides a potential scaffold for the development of new, broad-spectrum antibiotics. We used a structure-based approach to develop novel aryl ether analogues of triclosan that target ENR, the product of the fabI gene, from B. anthracis (BaENR). Structure-based design methods were used for the expansion of the compound series including X-ray crystal structure determination, molecular docking, and QSAR methods. Structural modifications were made to both phenyl rings of the 2-phenoxyphenyl core. A number of compounds exhibited improved potency against BaENR and increased efficacy against both the Sterne strain of B. anthracis and the methicillin-resistant strain of S. aureus. X-ray crystal structures of BaENR in complex with triclosan and two other compounds help explain the improved efficacy of the new compounds and suggest future rounds of optimization that might be used to improve their potency.

Effect of ring strain on the thiolate-disulfide exchange. A computational study.

B3LYP/aug-cc-pVDZ and MP2/6-31+G calculations of the reactions of HS(-) with small cyclic disulfides (dithiirane, 1,2-dithietane, 1,2-dithiolane, and 1,2-dithiane) were performed to determine the reaction mechanism. For the five- and six-membered rings, the reaction proceeds via the addition-elimination pathway, consistent with acyclic analogues. The smaller, more strained three- and four-membered rings react by the S(N)2 mechanism. Addition of the nucleophile cannot be accommodated by the small rings without concomitant ring cleavage.

Design, synthesis, and evaluation of oxyanion-hole selective inhibitor substituents for the S1 subsite of factor Xa.

We have designed, synthesized, and evaluated the factor Xa inhibitory activities of p-amidinophenyl-sulfones, amines, and alcohols intended to take advantage of the polarity and hydrogen-bonding potential of the oxyanion hole region of the S1 specificity pocket. We demonstrate that placement of an anionic group within the oxyanion hole region of the catalytic site substantially enhances activity, with small flexible groups favored over bulkier ones. Ab initio pKa calculations suggest that the hydroxyl substituent frequently used for benzamidine moieties may be ionized to form an anionic group, consistent with the general trend. One nonamidine based substituent also shows promising activity.