Molecular modeling of neurokinin B and tachykinin NK3 receptor complex.

The tachykinin receptor NK3 is a member of the rhodopsin family of G-protein coupled receptors. The NK3 receptor has been regarded as an important drug target due to diverse physiological functions and its possible role in the pathophysiology of psychiatric disorders, including schizophrenia. The NK3 receptor is primarily activated by the tachykinin peptide hormone neurokinin B (NKB) which is the most potent natural agonist for the NK3 receptor. NKB has been reported to play a vital role in the normal human reproduction pathway and in potentially life threatening diseases such as pre-eclampsia and as a neuroprotective agent in the case of neurodegenerative diseases. Agonist binding to the receptor is a critical event in initiating signaling, and therefore a characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKB with its G-protein coupled receptor NK3 has been developed. A three-dimensional model for the NK3 receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKB to the receptor has been performed utilizing limited ligand binding data obtained from photoaffinity labeling and site-directed mutagenesis studies. A molecular model for the NKB-NK3 receptor complex obtained sheds light on the topographical features of the binding pocket of the receptor and provides insight into the biochemical data currently available for the receptor.

Structural analysis and molecular modelling of the Cu/Zn-SOD from fungal strain Humicola lutea 103.

The native form of Cu/Zn-superoxide dismutase, isolated from fungal strain Humicola lutea 103 is a homodimer that coordinates one Cu(2+) and one Zn(2+) per monomer. Cu(2+) and Zn(2+) ions play crucial roles in enzyme activity and structural stability, respectively. It was established that HLSOD shows high pH and temperature stability. Thermostability of the glycosylated enzyme Cu/Zn-SOD, isolated from fungal strain H. lutea 103, was determined by CD spectroscopy. Determination of reversibility toward thermal denaturation for HLSOD allowed several thermodynamic parameters to be calculated. In this communication we report the conditions under which reversible denaturation of HLSOD exists. The narrow range over which the system is reversible has been determined using the strongest test of two important thermodynamic independent variables (T and pH). Combining both these variables, the "phase diagram" was determined, as a result of which the real thermodynamic parameters (ΔC(p), ΔH(exp)°, and ΔG(exp)°) was established. Because very narrow pH-interval of transitions we assume they are as result of overlapping of two simple transitions. It was found that ΔH(o) is independent from pH with a value of 1.3 kcal/mol and 2.8 kcal/mol for the first and the second transition, respectively. ΔG(o) was pH-dependent in all studied pH-interval. This means that the transitions are entropically driven, these. Based on this, these processes can be described as hydrophobic rearrangement of the quaternary structure. It was also found that glycosylation does not influence the stability of the enzyme because the carbohydrate chain is exposed on the surface of the molecule.

Molecular modeling of the peptide agonist-binding site in a neurokinin-2 receptor.

The neurokinin-2 receptor is a member of the rhodopsin family of G-protein coupled receptors, which represents one of the most relevant target families in small-molecule drug design. NK-2 receptors have been implicated in playing a pathophysiological role in asthma. Activation of the NK-2 receptor by its endogenous peptide agonist, tachykinins, is associated with diverse biological responses like bronchoconstriction, vasodepression, and regulation of endocrine functions. Agonist binding to the receptor is a crucial event in initiating signaling, and therefore characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKA with its G-protein coupled receptor neurokinin-2 receptor has been developed. A three-dimensional model for the NK-2 receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKA to the receptor has been performed utilizing the ligand binding data obtained from the photoaffinity labeling and site-directed mutagenesis studies. The molecular model for the NKA/NK-2 receptor complex thus obtained sheds light on the topographical features of the binding pocket of the receptor and provides atomic insight into the biochemical data currently available for the receptor. The results of the receptor modeling studies have been used to discuss the molecular determinants for NK-2 receptor selectivity.

Structure-based design of a potent and selective small peptide inhibitor of Mycobacterium tuberculosis 6-hydroxymethyl-7, 8-dihydropteroate synthase: a computer modelling approach.

In an attempt to design novel anti-TB drugs, the target chosen is the enzyme 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS), which is an attractive target since it is present in microorganisms but not in humans. The existing drugs for this target are the sulfa drugs, which have been used for about seven decades. However, single mutations in the DHPS gene can cause resistance to sulfa drugs. Therefore, there is a need for the design of novel drugs. Based on the recently determined crystal structure of Mycobacterium tuberculosis (M.tb) DHPS complexed with a known substrate analogue, and on the crystal structures of E. coli DHPS and Staphylococcus aureus DHPS, we have identified a dipeptide inhibitor with the sequence WK. Docking calculations indicate that this peptide has a significantly higher potency than the sulfa drugs. In addition, the potency is 70-90 times higher for M.tb DHPS as compared to that for the pterin and folate-binding sites of key human proteins. Thus, the designed inhibitor is a promising lead compound for the development of novel antimycobcaterial agents.

In silico structure-based design of a potent and selective small peptide inhibitor of protein tyrosine phosphatase 1B, a novel therapeutic target for obesity and type 2 diabetes mellitus: a computer modeling approach.

Protein Tyrosine Phosphatase 1B (PTP1B) has been shown to be a negative regulator of insulin signaling by dephosphorylating key tyrosine residues within the regulatory domain of the beta-subunit of the insulin receptor. Recent gene knockout studies in mice have shown the mice to have increased insulin sensitivity and improved glucose tolerance. Furthermore, these mice also exhibited a resistance to diet induced obesity. Inhibitors of PTP1B would have the potential of enhancing insulin action by prolonging the phosphorylated state of the insulin receptor. In addition, recent clinical studies have shown that the haplotype ACTTCAG0 of the PTPN1 gene, which encodes PTP1B, is a major risk contributor to type 2 diabetes mellitus (T2DM). Thus, there is compelling evidence that small molecule inhibitors of PTP1B may be effective in treating insulin resistance at an early stage, thereby leading to a prevention strategy for T2DM and obesity. Based on the crystal structure of the complex of PTP1B with a known inhibitor, we have identified a tetrapeptide inhibitor with the sequence WKPD. Docking calculations indicate that this peptide is as potent as the existing inhibitors. Moreover, the peptide is also found to be selective for PTP1B with a greatly reduced potency against other biologically important protein tyrosine phosphatases such as PTP-LAR, Calcineurin, and the highly homologous T-Cell Protein Tyrosine Phosphatase (TCPTP). Thus the designed tetrapeptide is a suitable lead compound for the development of new drugs against type 2 diabetes and obesity.

In silico structure-based design of a potent, mutation resilient, small peptide inhibitor of HIV-1 reverse transcriptase.

A crucial step in the replication of HIV-1 is the conversion of its single-stranded RNA to double-stranded DNA, which is catalyzed by the virally encoded reverse transcriptase (RT). The latter is therefore a key target for the development of anti-HIV drugs. Currently approved anti-RT drugs fall into two main classes: (i). nucleoside analog inhibitors which are incorporated into the primer strand in their metabolically activated triphosphate forms, causing termination of DNA synthesis due to their 3'-deoxy configuration and (ii). the non-nucleoside inhibitors (NNIs), which are generally specific for HIV-1 RT and bind at an allosteric site approximately 10 A from the active site causing a displacement of the catalytic aspartate residues. The so-called "first generation" NNI drugs are generally susceptible to the effects of single-point mutations within RT, while more recent "second generation" NNIs, such as efavirenz, the carboxanilide UC-781 and certain quinoxalines demonstrate much greater resilience to mutations in RT. The crystal structures of the complexes of wild type and mutant RTs with first and second generation NNIs have shown that, for an inhibitor to be potent as well as mutation resilient, it should (i). make hydrogen bonds with the main chain of RT, (ii). have a large number of interactions with RT and (iii). have the ability to rearrange and adapt to a mutated NNI pocket. Based on the crystal structures of the complexes of wild type RT and Tyr188Cys mutant of RT with UC-781, we have designed a small peptide inhibitor. Docking results on this peptide using AutoDock3.0 and SYBYL 6.8.1 indicate that the peptide has a potency comparable to that of UC-781 with a retention of activity against the Tyr188Cys mutant RT. The proposed, small peptide is seen to possess all the desirable features of a potent and mutation resilient inhibitor and is hence a potential lead compound.

Structure-based design of a novel peptide inhibitor of HIV-1 integrase: a computer modeling approach.

The insertion of viral DNA into the host chromosome is an essential step in the replication of HIV-1, and is carried out by an enzyme, HIV-1 integrase (IN). Since the latter has no human cellular counterpart, it is an attractive target for antiviral drug design. Several IN inhibitors having activities in the micromolar range have been reported to date. However, no clinically useful inhibitors have yet been developed. Recently reported diketo acids represent a novel and selective class of IN inhibitors. These are the only class which appear to selectively target integrase and two of the inhibitors, L-708,906 and L-731,988, are the most potent inhibitors of preintegration complexes described to date. The X-ray crystal structure of the IN catalytic domain complexed with a diketo acid derivative inhibitor, 5CITEP, has recently been determined. Although the structure is of great value as a platform for drug design, experimental data suggest that crystal packing effects influence the observed inhibitor position. This has been confirmed by computational docking studies using the latest version (3.0) of the AutoDock program, which has been shown to give results largely consistent with available experimental data. Using AutoDock 3.0 and SYBYL6.6 we have modeled the complexes of IN with the diketo acid inhibitors so as to identify the enzyme binding site. In the quest for novel, potent and selective small molecule inhibitors, we present here a new approach to peptide inhibitor design using a, b- unsaturated (dehydro) residues, which confer a unique conformation on a peptide sequence. Based on the above models, we selected a tetrapeptide sequence containing a dehydro-Phe residue, which was found to have an open conformation as ascertained from its X-ray crystal structure. Docking results on this peptide led us to propose a modification at the C-terminal end. The modified peptide was found to dock in a similar position as the diketo acid inhibitors and was predicted to have a comparable potency.