Using a structure based pharmacophore design, a weak inhibitor of RNase H, identified from a small library of two metal binding HIV-1 integrase inhibitors, was optimized for potency and physicochemical properties. This manuscript describes the SAR and in vivo DMPK for the pyridopyrimidinone class of inhibitors.

Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir's antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir's prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.

ABSTRACT The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10 −6 s −1 and a dissociative half-life ( t 1/2 ) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding ( t 1/2 , at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

Many proteins involved in signal transduction and protein trafficking are posttranslationally modified by the covalent attachment of lipid groups. One form of lipid modification involves attachment of either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid lipid to a cysteine residue fourth from the C-terminus of the substrate protein. The attachment of the isoprenoid is the first step in a processing pathway that can include subsequent proteolysis of three carboxyl-terminal residues, methylation of the free carboxyl group of the resulting C-terminal prenylcysteine, and modification with additional lipid molecules. These modifications are necessary for targeting and attachment of these so-called CAAX proteins to the correct membrane as well as for the cellular function of the protein. The focus of this chapter is on the two protein prenyltransferases responsible for addition of the isoprenoid to the CAAX protein substrates.

A novel class of 3,7-diphenyl-4-amino-thieno and furo[3,2-c]pyridine has been designed based on pharmacophore models of ATP competitive kinase inhibitors. Versatile synthetic methods via double Suzuki coupling to explore SAR have been established and potent inhibitors against angiogenetic targets, VEGFR2, Tie-2, and EphB4, have been successfully discovered.

The identification and exploration of a novel, potent and selective series of N-(3-cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)amide inhibitors of JNK2 and JNK3 kinases is described. Compounds 5a and 11a were identified as potent inhibitors of JNK3 (pIC50 6.7 and 6.6, respectively), with essentially equal potency against JNK2 (pIC50 6.5). Selectivity within the mitogen-activated protein kinase (MAPK) family, against JNK1, p38alpha and ERK2, was observed for the series. X-ray crystallography of 5e and 8a in JNK3 revealed a unique binding mode, with the 3-cyano substituent forming an H-bond acceptor interaction with the hinge region of the ATP-binding site.

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the farnesylation of a cysteine residue of protein or peptide substrates containing the CaaX motif using farnesyl pyrophosphate (FPP) [1], FTase has been hypothesized to utilize an electrophilic mechanism (Fig. 1, left) because of a decrease in catalysis observed upon the addition of electron-withdrawing groups on the FPP C1 carbon [2]. However, consistent with a nucleophilic mechanism (Fig. 1, right), a direct metal ion-sulfur bond has been detected in absorbance spectra of cobalt-substituted FTase [3]. Also, the binding affinity of GCVLS, a peptide derived from the C-terminus of H-Ras, was recently shown to be dependent upon pH [4] suggesting that the of the cysteine thiol was lowered from 8.1 (free) to 6.4 (bound). The formation of a bound thiolate at physiological pH suggests that the catalytic rate might be enhanced by an increase in thiolate concentration. Substitution of a more thiophilic metal in place of zinc should lower the peptide thiol even further.

Zinc metalloenzymes catalyze many important cellular reactions. Recently, the involvement of zinc in the catalysis of alkylation of sulfur groups has gained prominence. Current studies of the zinc metalloenzyme protein farnesyltransferase have shed light on its structure and catalytic mechanism, as well as the general mechanism of zinc-catalyzed sulfur alkylation.

The catalytic portion (F1) of ATP synthases have the subunit composition α3, β3, γ, δ, ε. This composition imparts structural asymmetry to the entire complex that results in differences in nucleotide binding affinity among the six binding sites. Evidence that two or more sites participate in catalysis, alternating their properties, led to the notion that the interactions of individual αβ pairs with the small subunits must change as binding site properties alternate. A rotation of the γ subunit within the α3β3 hexamer has been proposed as a means of alternating the properties of catalytic sites. Evidence argues that the rotation of the complete γ subunit during ATP hydrolysis is not mandatory for activity. The γ subunit of chloroplast F1 may be cleaved into three large fragments that remain bound to F1. This cleavage enhances ATPase activity without loss of evidence of site-site interactions. Complexes of α3β3 have been shown to have significant ATPase activity in the absence of γ. Mg2+ATP affects the interaction of γ with the different β subunits, and induces other changes in F1, but whether these changes are induced by catalysis, or are fast enough to be involved in the catalytic turnover of the enzyme has not been established. Likewise, changes in structure and in binding site properties induced in thylakoid membrane bound CF1 by formation of an electrochemical proton gradient may activate the enzyme rather than be apart of catalysis. Mechanisms other than rotary catalysis should be considered.