Inhibitors directed against two distinct points in the HIV-1 life cycle are being prepared. These inhibitors are intended to serve as potential new therapeutics and as pharmacological probes to investigate biochemical mechanisms of viral replication. The two areas of investigation are: (1) HIV-1 integrase (IN), where inhibitors may disrupt incorporation of viral cDNA into the host genome;(2) Binding of HIV p6Gag protein to human Tsg101 protein, where inhibitors may disrupt viral assembly and budding (1) HIV-1 IN inhibitors: This work is being done in collagoration with Drs. Yves Pommier (CCR, NCI) Steven Hughes (CCR, NCI) and Peter Cherepanov (Imperial College, London). A large number of IN inhibitors have been reported. Many of these exhibit common key structural features. These features include a co-planar arrangement of heteroatoms that chelate magnesium ions. Halogen-substituted aromatic functionality linked to the chelating portion of the inhibitors has also been shown to interact with a region formed between a viral DNA base and the protein in the IN-DNA complex. This class of IN inhibitors is thought to function by chelating Mg2+ ions within the IN catalytic site, where they selectively inhibit strand transfer (ST) reactions over 3-processing (3-P) reactions. We have developed 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one and 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones, which are structurally simple IN inhibitors that exhibit good potency and ST selectivity in vitro in the presence of Mg2+ cofactor. Our efforts to develop new inhibits based on these analogues are being guided by co-crystal structures of our 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones bound to the IN-DNA complex of the primitive foamy virus (PFV) integrase (done in collaboration with Dr. Peter Cherepanov (Imperial Colleage, London). Most recently, we have prepared sulfonamide-containing variants of the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one platform that exhibit low nanomolar IC50 values against HIV-1 IN in in vitro assays, and in antiviral assays employing viral vectors. These data compare favorably to Raltegravir, the only current FDA-aproved HIV IN inhibitor. However, the potential therapeutic utility of these compounds is limited by unacceptable cytotoxicity that may arise from embedded catechol functionality. In order to remove this catechol functionality, we designed a series of hydroxy-pyrrolopyridine-trione-containing analogues. The efficient syntheses of these compounds relies on the application of Pummerer cyclization deprotonation cycloaddition cascades of imidosulfoxides as well as [3+2] cycloadditions of isomnchnones. Although the potency of these inhibitors was not as great as the original 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-ones, a representative inhibitor retained most of its inhibitory potency against the three major raltegravir-resistance mutant IN enzymes, G140S/Q148H, Y143R and N155H. In antiviral assays employing viral vectors coding these IN mutants, compound this compound was approximately 200-fold and 20-fold less affected than raltegravir against the G140S/Q148H and Y143R mutations, respectively. Against the N155H mutation the conpound was approximately 10-fold less affected than raltegravir. This latter series of compounds represent a novel structural class that may be further developed to overcome resistance to raltegravir, particularly in the case of the G140S/Q148H mutations. (2) Tsg101-binding inhibitors: Binding of the HIV p6Gag protein to human Tsg101 protein has been shown to be necessary for viral budding and to involve a critical 9-mer P-E-P-T-A-P-P-E-E sequence of the p6 protein. In a collaboration with Dr. Eric Freed (CCR, NCI, NIH) we are preparing peptide and peptide mimetic variants of this 9-mer sequence as Tsg101-binding antagonists that may lead to a new class of viral budding inhibitors. A unifying principal guiding our approach is the incorporation of amino-oxy functionality into peptide residues that can be easily functionalized in a single step to provide a library of oxime derivatives. This approach has resulted in the identification of several low micromolar affinity Tsg101 binding antagonists. Further oxime library diversification is being guided by in silico modeling based on a co-crystal structure of our high affinity inhibitor bound to Tsg101 protein. This structure was obtained through a collaboration with Dr. James Hurley (NIDDK, NIH). Fluorescent labeling has been introduced into one of the high affinity-binding antagonists to produce a reagent that will be used for identification of Tsg101-binding antagonists by high throughput screening of small compound libraries in the NIH Chemical Genomics Center under the direction of Dr. Doug Auld. In parallel work, the chemical biology of Tsg101 interactions with peptide ligands is being explored using conformationally-constrained peptide macrocycles. These were formed by ring-closing metathesis reactions on peptide-peptoid precursors. These macrocycles exhibit higher Tsg101-binding affinity and better cellular bioavailability than the parent open-chain peptides.
Showing the most recent 10 out of 20 publications
