The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.
Proteochemometrics was applied in the analysis of the binding of organic compounds to wild-type and chimeric melanocortin receptors. Thirteen chimeric melanocortin receptors were designed based on statistical molecular design; each chimera contained parts from three of the MC 1,3-5 receptors. The binding affinities of 18 compounds were determined for these chimeric melanocortin receptors and the four wild-type melanocortin receptors. The data for 14 of these compounds were correlated to the physicochemical and structural descriptors of compounds, binary descriptors of receptor sequences, and cross-terms derived from ligand and receptor descriptors to obtain a proteochemometric model (correlation was performed using partial least-squares projections to latent structures; PLS). A well fitted mathematical model (R 2 ϭ 0.92) with high predictive ability (Q 2 ϭ 0.79) was obtained. In a further validation of the model, the predictive ability for ligands (Q 2 lig ϭ 0.68) and receptors (Q 2 rec ϭ 0.76) was estimated. The model was moreover validated by external prediction by using the data for the four additional compounds that had not at all been included in the proteochemometric model; the analysis yielded a Q 2 ext ϭ 0.73. An interpretation of the results using PLS coefficients revealed the influence of particular properties of organic compounds on their affinity to melanocortin receptors. Threedimensional models of melanocortin receptors were also created, and physicochemical properties of the amino acids inside the receptors' transmembrane cavity were correlated to the PLS modeling results. The importance of particular amino acids for selective binding of organic compounds was estimated and used to outline the ligand recognition site in the melanocortin receptors.Melanocortin receptors (MCRs) are members of the seven transmembrane (TM)-spanning G protein-coupled receptor (GPCR) superfamily. To date, five MCR subtypes, MC 1-5, are recognized in mammals, and each of these subtypes stimulates cAMP signal transduction pathways. An endogenous group of peptides, the melanocyte-stimulating hormones (MSH) and corticotropin and agouti and agouti-related protein, bind to the MCRs with agonistic and antagonistic properties, respectively. However, an exception is the MC 2 R, which binds only the corticotropin (Schioth et al., 1996a).The MCRs have a wide range of physiological roles. The MC 1 R regulates melanin pigment formation in the skin and has a regulating role in the immune system. The MC 2 R regulates corticosteroid production of the adrenals. The MC 3 and MC 4 Rs play important roles in controlling feeding and sexual behaviors, and the MC 5 R is involved in the regulation of exocrine glands (Wikberg et al., 2000Wikberg, 2001). The potential of using the MCRs as targets for drugs to treat important medical conditions such as obesity/diabetes, inflammatory conditions, and sexual dysfunctions prompts the need for compounds that show high specificity for particular MCR subtypes. However, the design of selective dru...
A series of 45 peptide inhibitors was designed, synthesized, and evaluated against the NS2B-NS3 proteases of the four subtypes of dengue virus, DEN-1-4. The design was based on proteochemometric models for Michaelis (Km) and cleavage rate constants (kcat) of protease substrates. This led first to octapeptides showing submicromolar or low micromolar inhibitory activities on the four proteases. Stepwise removal of cationic substrate non-prime side residues and variations in the prime side sequence resulted finally in an uncharged tetrapeptide, WYCW-NH2, with inhibitory Ki values of 4.2, 4.8, 24.4, and 11.2 μM for the DEN-1-4 proteases, respectively. Analysis of the inhibition data by proteochemometric modeling suggested the possibility for different binding poses of the shortened peptides compared to the octapeptides, which was supported by results of docking of WYCW-NH2 into the X-ray structure of DEN-3 protease.
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