Structural and biochemical analysis of human inositol monophosphatase-1 inhibition by ebselen.

Bipolar disorder is a major psychiatric disorder associated with cognitive impairment and a high suicide rate. Frontline therapy for this condition includes lithium (Li+)-containing treatments that can exert severe side effects. One target of Li+ is inositol monophosphatase-1 (IMPase1); inhibition of IMPase1 through small-molecule compounds may provide an alternative treatment for bipolar disorder. One such compound is the anti-inflammatory drug ebselen, which is well tolerated and safe; however, ebselen's exact mechanism of action in IMPase1 inhibition is not fully understood, preventing rational design of IMPase1 inhibitors. To fill this gap, we performed crystallographic and biochemical studies to investigate how ebselen inhibits IMPase1. We obtained a structure of IMPase1 in space group P21 after treatment with ebselen that revealed three key active-site loops (residues 33-44, 70-79, and 161-165) that are either disordered or in multiple conformations, supporting a hypothesis whereby dynamic conformational changes may be important for catalysis and ebselen inhibition. Using the thermal shift assay, we confirmed that ebselen significantly destabilizes the enzyme. Molecular docking suggests that ebselen could bind in the vicinity of His217. Investigation of the role of IMPase1 residues His217 and Cys218 suggests that inhibition of IMPase1 by ebselen may not be mediated via covalent modification of the active-site cysteine (Cys218) and is not affected by the covalent modification of other cysteine residues in the structure. Our results suggest that effects previously ascribed to ebselen-dependent inhibition likely result from disruption of essential active-site architecture, preventing activation of the IMPase1-Mg2+ complex.Communicated by Ramaswamy H. Sarma.

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima.

Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine-bound structure indicates that it consists of a closed monomer with its C-terminal helix folded against the rest of the protein, as typically observed for substrate-binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.

Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CLpro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CLpro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CLpro . HKU4-CoV 3CLpro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CLpro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CLpro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CLpro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CLpro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CLpro . HIGHLIGHTS: MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CLpro is a potential drug target for coronavirus infection. HKU4-CoV 3CLpro is a useful surrogate model for the identification of MERS-CoV 3CLpro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CLpro inhibitors development.

Innovative computer-aided methods for the discovery of new kinase ligands.

Recent evidence points to significant roles played by protein kinases in cell signaling and cellular proliferation. Faulty protein kinases are involved in cancer, diabetes and chronic inflammation. Efforts are continuously carried out to discover new inhibitors for selected protein kinases. In this review, we discuss two new computer-aided methodologies we developed to mine virtual databases for new bioactive compounds. One method is ligand-based exploration of the pharmacophoric space of inhibitors of any particular biotarget followed by quantitative structure-activity relationship-based selection of the best pharmacophore(s). The second approach is structure-based assuming that potent ligands come into contact with binding site spots distinct from those contacted by weakly potent ligands. Both approaches yield pharmacophores useful as 3D search queries for the discovery of new bioactive (kinase) inhibitors.

Exploration of piperidinols as potential antitubercular agents.

Novel drugs to treat tuberculosis are required and the identification of potential targets is important. Piperidinols have been identified as potential antimycobacterial agents (MIC < 5 µg/mL), which also inhibit mycobacterial arylamine N-acetyltransferase (NAT), an enzyme essential for mycobacterial survival inside macrophages. The NAT inhibition involves a prodrug-like mechanism in which activation leads to the formation of bioactive phenyl vinyl ketone (PVK). The PVK fragment selectively forms an adduct with the cysteine residue in the active site. Time dependent inhibition of the NAT enzyme from Mycobacterium marinum (M. marinum) demonstrates a covalent binding mechanism for all inhibitory piperidinol analogues. The structure activity relationship highlights the importance of halide substitution on the piperidinol benzene ring. The structures of the NAT enzymes from M. marinum and M. tuberculosis, although 74% identical, have different residues in their active site clefts and allow the effects of amino acid substitutions to be assessed in understanding inhibitory potency. In addition, we have used the piperidinol 3-dimensional shape and electrostatic properties to identify two additional distinct chemical scaffolds as inhibitors of NAT. While one of the scaffolds has anti-tubercular activity, both inhibit NAT but through a non-covalent mechanism.

Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages.

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs.

Comparison of the Arylamine N-acetyltransferase from Mycobacterium marinum and Mycobacterium tuberculosis.

Arylamine N-acetyltansferase (NAT) from Mycobacterium tuberculosis (TBNAT) is a potential drug target for anti-tubercular therapy. Recombinant TBNAT is much less soluble and is produced in lower yields than the closely related NAT from Mycobacterium marinum (MMNAT). In order to explore MMNAT as a model for TBNAT in drug discovery, we compare the two mycobacterial NAT enzymes. Two site-directed mutants of MMNAT have been prepared and characterised: MMNAT71, Tyr --> Phe and MMNAT209, Met --> Thr, in which residues within 6 A of the active-site cysteine have been replaced with the corresponding residue from TBNAT. Two chimeric proteins have also been produced in which the third domain of MMNAT has been replaced by the third domain of TBNAT and vice versa. The activity profile of the chimeric proteins suggests a role for the third domain in the evolutionary divergence of NAT between these closely related mycobacterial species.