ChemoDOTS: a web server to design chemistry-driven focused libraries.

In drug discovery, the successful optimization of an initial hit compound into a lead molecule requires multiple cycles of chemical modification. Consequently, there is a need to efficiently generate synthesizable chemical libraries to navigate the chemical space surrounding the primary hit. To address this need, we introduce ChemoDOTS, an easy-to-use web server for hit-to-lead chemical optimization freely available at https://chemodots.marseille.inserm.fr/. With this tool, users enter an activated form of the initial hit molecule then choose from automatically detected reactive functions. The server proposes compatible chemical transformations via an ensemble of encoded chemical reactions widely used in the pharmaceutical industry during hit-to-lead optimization. After selection of the desired reactions, all compatible chemical building blocks are automatically coupled to the initial hit to generate a raw chemical library. Post-processing filters can be applied to extract a subset of compounds with specific physicochemical properties. Finally, explicit stereoisomers and tautomers are computed, and a 3D conformer is generated for each molecule. The resulting virtual library is compatible with most docking software for virtual screening campaigns. ChemoDOTS rapidly generates synthetically feasible, hit-focused, large, diverse chemical libraries with finely-tuned physicochemical properties via a user-friendly interface providing a powerful resource for researchers engaged in hit-to-lead optimization.

Genetic, structural, and chemical insights into the dual function of GRASP55 in germ cell Golgi remodeling and JAM-C polarized localization during spermatogenesis.

Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.

Crystal structure of the vaccinia virus DNA polymerase holoenzyme subunit D4 in complex with the A20 N-terminal domain.

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase E9, the uracil-DNA glycosylase D4 and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase co-factor whose function is essential for processive DNA synthesis. Genetic and biochemical data have established that residues located in the N-terminus of A20 are critical for binding to D4. However, no information regarding the residues of D4 involved in A20 binding is yet available. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A201₋50). We showed that whereas D4 forms homodimers in solution when expressed alone, D4/A201₋50 clearly behaves as a heterodimer. The crystal structure of D4/A201₋50 solved at 1.85 Å resolution reveals that the D4/A20 interface (including residues 167 to 180 and 191 to 206 of D4) partially overlaps the previously described D4/D4 dimer interface. A201₋50 binding to D4 is mediated by an α-helical domain with important leucine residues located at the very N-terminal end of A20 and a second stretch of residues containing Trp43 involved in stacking interactions with Arg167 and Pro173 of D4. Point mutations of the latter residues disturb D4/A201₋50 formation and reduce significantly thermal stability of the complex. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A201₋50 interaction. Finally, we propose a model of D4/A201₋50 in complex with DNA and discuss a number of mutants described in the literature, which affect DNA synthesis. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase cofactor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface.

Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.

2P2Idb: a structural database dedicated to orthosteric modulation of protein-protein interactions.

Protein-protein interactions are considered as one of the next generation of therapeutic targets. Specific tools thus need to be developed to tackle this challenging chemical space. In an effort to derive some common principles from recent successes, we have built 2P2Idb (freely accessible at http://2p2idb.cnrs-mrs.fr), a hand-curated structural database dedicated to protein-protein interactions with known orthosteric modulators. It includes all interactions for which both the protein-protein and protein-ligand complexes have been structurally characterized. A web server provides links to related sites of interest, binding affinity data, pre-calculated structural information about protein-protein interfaces and 3D interactive views through java applets. Comparison of interfaces in 2P2Idb to those of representative datasets of heterodimeric complexes has led to the identification of geometrical parameters and residue properties to assess the druggability of protein-protein complexes. A tool is proposed to calculate a series of biophysical and geometrical parameters that characterize protein-protein interfaces. A large range of descriptors are computed including, buried accessible surface area, gap volume, non-bonded contacts, hydrogen-bonds, atom and residue composition, number of segments and secondary structure contribution. All together the 2P2I database represents a structural source of information for scientists from academic institutions or pharmaceutical industries.

Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.

The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90ß-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

PDZome-wide and structural characterization of the PDZ-binding motif of VANGL2.

VANGL2 is a core component of the non-canonical Wnt/Planar Cell Polarity signaling pathway that uses its highly conserved carboxy-terminal type 1 PDZ-binding motif (PBM) to bind a variety of PDZ proteins. In this study, we characterize and quantitatively assess the largest VANGL2 PDZome-binding profile documented so far, using orthogonal methods. The results of our holdup approach support VANGL2 interactions with a large panel of both long-recognized and unprecedented PDZ domains. Truncation and point mutation analyses of the VANGL2 PBM establish that, beyond the strict requirement of the P-0 / V521 and P-2 / T519 amino acids, upstream residues, including E518, Q516 and R514 at, respectively, P-3, P-5 and P-7 further contribute to the robustness of VANGL2 interactions with two distinct PDZ domains, SNX27 and SCRIBBLE-PDZ3. In agreement with these data, incremental amino-terminal deletions of the VANGL2 PBM causes its overall affinity to progressively decline. Moreover, the holdup data establish that the PDZome binding repertoire of VANGL2 starts to diverge significantly with the truncation of E518. A structural analysis of the SYNJ2BP-PDZ/VANGL2 interaction with truncated PBMs identifies a major conformational change in the binding direction of the PBM peptide after the P-2 position. Finally, we report that the PDZome binding profile of VANGL2 is dramatically rearranged upon phosphorylation of S517, T519 and S520. Our crystallographic approach illustrates how SYNJ2BP accommodates a S520-phosphorylated PBM peptide through the ideal positioning of two basic residues, K48 and R86. Altogether our data provides a comprehensive view of the VANGL2 PDZ network and how this network specifically responds to the post-translation modification of distinct PBM residues. These findings should prove useful in guiding future functional and molecular studies of the key PCP component VANGL2.

Functional consequence of covalent reaction of phosphoenolpyruvate with UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA).

The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this "dormant" complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.

Stereoselective synthesis of original spirolactams displaying promising folded structures.

Access to diastereoisomeric forms of original spirolactam frameworks and investigation of their folded potentials are depicted here. Taking advantage of a stereoselective ring-contraction reaction, the Transannular Rearrangement of Activated Lactams (TRAL), followed by two unprecedented tandem reactions, we describe here an efficient access to elegant spirocyclic scaffolds. After dimerization, NMR analyses, circular dichroism, SEM and molecular modelling indicated the existence of an attractive edifice able to fold and behave as a PPII helix, a common yet neglected peptidic secondary structure.

Discovery of a PDZ Domain Inhibitor Targeting the Syndecan/Syntenin Protein-Protein Interaction: A Semi-Automated "Hit Identification-to-Optimization" Approach.

The rapid identification of early hits by fragment-based approaches and subsequent hit-to-lead optimization represents a challenge for drug discovery. To address this challenge, we created a strategy called "DOTS" that combines molecular dynamic simulations, computer-based library design (chemoDOTS) with encoded medicinal chemistry reactions, constrained docking, and automated compound evaluation. To validate its utility, we applied our DOTS strategy to the challenging target syntenin, a PDZ domain containing protein and oncology target. Herein, we describe the creation of a "best-in-class" sub-micromolar small molecule inhibitor for the second PDZ domain of syntenin validated in cancer cell assays. Key to the success of our DOTS approach was the integration of protein conformational sampling during hit identification stage and the synthetic feasibility ranking of the designed compounds throughout the optimization process. This approach can be broadly applied to other protein targets with known 3D structures to rapidly identify and optimize compounds as chemical probes and therapeutic candidates.

From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia.

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

A novel approach to the discovery of small-molecule ligands of CDK2.

In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 µM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.

Antimicrobial and cytotoxic effects of marine sponge extracts Agelas clathrodes, Desmapsamma anchorata and Verongula rigida from a Caribbean Island.

Although marine sponges are known for their antimicrobial, antifungal and cytotoxic activity, very few studies have been carried out on endemic species of Martinique. Martinique is part of the Agoa Sanctuary, a marine protected area that includes the exclusive economic zones (EEZ) of the French Caribbean islands, making it an abundant source of marine species. To highlight the potential of this area for the discovery of marine biomolecules with antipathogenic and antitumor activities, we tested the aqueous and ethanolic extracts of sponge species Agelas clathrodes, Desmapsamma anchorata and Verongula rigida. Five bacterial strains: Bacillus cereus (CIP 78.3), Escherichia coli (CIP 54.127), Pseudomonas aeruginosa (CIP A22), Staphylococcus aureus (CIP 67.8) and Staphylococcus saprophyticus (CIP 76125) were evaluated, as well as four tumor cell lines: breast cancer (MDA-MB231), glioblastoma (RES259) and leukemia (MOLM14 and HL-60). Antimicrobial activity was evaluated using the disc diffusion technique by determining the minimum inhibitory and minimum bactericidal concentrations. Tumor cytotoxic activity was determined in vitro by defining the minimum concentration of extracts that would inhibit cell growth. Ethanolic extracts of Agelas clathrodes were bactericidal for Staphylococcus aureus and Staphylococcus saprophyticus strains, as well as strongly cytotoxic (IC50 < 20 µg/mL) on all cancer cell lines. Verongula rigida also showed strong cytotoxic activity on cell lines but no antimicrobial activity. These results are innovative for this species on these bacterial lines, highlighting the potential of sponge extracts from this area as bioactive compounds sources.

Discovery of Small-Molecule Inhibitors of the PTK7/ß-Catenin Interaction Targeting the Wnt Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC), the second cause of death due to cancer worldwide, is a major public health issue. The discovery of new therapeutic targets is thus essential. Pseudokinase PTK7 intervenes in the regulation of the Wnt/ß-catenin pathway signaling, in part, through a kinase domain-dependent interaction with the ß-catenin protein. PTK7 is overexpressed in CRC, an event associated with metastatic development and reduced survival of nonmetastatic patients. In addition, numerous alterations have been identified in CRC inducing constitutive activation of the Wnt/ß-catenin pathway signaling through ß-catenin accumulation. Thus, targeting the PTK7/ß-catenin interaction could be of interest for future drug development. We have developed a NanoBRET screening assay recapitulating the interaction between PTK7 and ß-catenin to identify compounds able to disrupt this protein-protein interaction. A high-throughput screening allowed us to identify small-molecule inhibitors targeting the Wnt pathway signaling and inducing antiproliferative and antitumor effects in vitro in CRC cells harboring ß-catenin or adenomatous polyposis coli (APC) mutations. Thus, inhibition of the PTK7/ß-catenin interaction could represent a new therapeutic strategy to inhibit cell growth dependent on the Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

Revisiting the Molecular Interactions between the Tumor Protein TCTP and the Drugs Sertraline/Thioridazine.

TCTP protein is a pharmacological target in cancer and TCTP inhibitors such as sertraline have been evaluated in clinical trials. The direct interaction of TCTP with the drugs sertraline and thioridazine has been reported in vitro by SPR experiments to be in the ∼30-50 µM Kd range (Amson et al. Nature Med 2012), supporting a TCTP-dependent mode of action of the drugs on tumor cells. However, the molecular details of the interaction remain elusive although they are crucial to improve the efforts of on-going medicinal chemistry. In addition, TCTP can be phosphorylated by the Plk-1 kinase, which is indicative of poor prognosis in several cancers. The impact of phosphorylation on TCTP structure/dynamics and binding with therapeutical ligands remains unexplored. Here, we combined NMR, TSA, SPR, BLI and ITC techniques to probe the molecular interactions between TCTP with the drugs sertraline and thioridazine. We reveal that drug binding is much weaker than reported with an apparent ∼mM Kd and leads to protein destabilization that obscured the analysis of the published SPR data. We further demonstrate by NMR and SAXS that TCTP S46 phosphorylation does not promote tighter interaction between TCTP and sertraline. Accordingly, we question the supported model in which sertraline and thioridazine directly interact with isolated TCTP in tumor cells and discuss alternative modes of action for the drugs in light of current literature.

CRCM5484: A BET-BDII Selective Compound with Differential Anti-leukemic Drug Modulation.

Differentially screening the Fr-PPIChem chemical library on the bromodomain and extra-terminal (BET) BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII-selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the bromodomain (BD) cavity and its potential as a starting scaffold for the development of potent and selective bromodomain inhibitors.

Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2'-O-methyltransferase (2'O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2'O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

The fungal product terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) .

Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus. We found that terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme MurA from Enterobacter cloacae and Escherichia coli in vitro. The crystal structure of the MurA dead-end complex with terreic acid revealed that the quinine ring is covalently attached to the thiol group of Cys115, the molecular target of the antibiotic fosfomycin. Kinetic characterization established that the inactivation requires the presence of substrate UNAG (UDP-N-acetylglucosamine), proceeding with an inactivation rate constant k(inact) of 130 M(-1) s(-1). Although the mechanisms of inactivation are similar, fosfomycin is approximately 50 times more potent than terreic acid, and the structural consequences of covalent modification by these two inhibitors are fundamentally different. The MurA-fosfomycin complex exists in the closed enzyme conformation, with the Cys115-fosfomycin adduct buried in the active site. In contrast, the dead-end complex with terreic acid is open, is free of UNAG, and has the Cys115-terreic acid adduct solvent-exposed. It appears that terreic acid reacts with Cys115 in the closed, binary state of the enzyme, but that the resulting Cys115-terreic acid adduct imposes steric clashes in the active site. As a consequence, the loop containing Cys115 rearranges, the enzyme opens, and UNAG is released. The differential kinetic and structural characteristics of MurA inactivation by terreic acid and fosfomycin reflect the importance of noncovalent binding potential, even for covalent inhibitors, in ensuring inactivation efficiency and specificity.

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity.

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF's tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein's flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC50 values lower than 10 microM (three hits with IC50 lower than 1 microM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC50=300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF's tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.