Inhibitors directed against three distinct points in the human immunodeficiency virus type 1 (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 three 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; and 3) binding of human cytidine deaminase APOBEC3G (A3G) to the HIV-1 protein Vif, where inhibitors may result in hyper-mutation of proviral DNA. 1) HIV-1 IN Inhibitors. Although a large number of inhibitors have been reported in the literature, diketoacid (DKA)-derived agents have recently shown particular promise, with members of this genre showing significant promise in anti-acquired immunodeficiency syndrome (AIDS) clinical trials. These agents are thought to function by chelating Mg2+ ions at the IN catalytic site, where they selectively inhibit strand transfer (ST) reactions over 3-processing (3-P) reactions. We had previously reported the bis-salicylhydrazides class of IN inhibitors that we also hypothesized function by metal chelation. However, members of this series exhibit potent inhibition only when Mn2+ is used as a cofactor and not when the physiologically-relevant Mg2+ is used. Our recent findings have shown that bis-aroylhydrazides could acquire inhibitory potency in the presence of Mg2+ through the inclusion of dihdroxybenzoyl substituents. Good selectivity for IN-catalyzed ST versus 3-P reactions could also be achieved using a 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one ring system as a conformationally-constrained 2,3-dihydroxybenzoyl equivalent. Adding a second oxo-group to the isoindole-1-one ring system gave the phthalimide-based isoindole-1,3(2H)-diones, which generally exhibited enhanced of 3-P and ST inhibitory potencies. Antiviral effects in cultured cells using HIV-1 based vectors showed that certain bicyclic conformationally constrained analogues exhibited sub-micromolar antiviral potencies against HIV-1 infected cells. Synthesis of a large number of structural variations has been undertaken to identify N-substituents that provide the highest efficacy. This work indicates that the 4,5-dihydroxyphthalimide nucleus offers a structurally simple starting point for the further development of IN inhibitors. 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. 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. One approach was to replace the Pro4 residue with N-substitued glycine (NSG) residues (termed peptoids). However, this is synthetically problematic. Therefore, we resorted to a new family of peptoid variants that incorporate hydrazone amides as NSG surrogates. These can be prepared readily in library fashion by reacting a series of aldehydes with a single high-performance liquid chromatography (HPLC)-purified hydrazide precursor following cleavage from the solid-phase resin. Reduction of these hydrazones to N-substitued peptoid hydrazides affords a facile route to library diversification. We have extended these studies by replacing several key residues in the parent p6-derived 9-mer sequence with other non-natural amino acid analogues. A unifying principle guiding our approach has been the incorporation of amino-oxy functionality into the residues that can be functionalized in a single final step to provide a library of oxime derivatives. This approach has resulted in the identification of several low micromolar affinity Tsg101 binding antagonists. Ongoing optimization of binding affinity is being continued by a variety of means, including macrocyclization using ring-closing metathesis (RCM) chemistries. X-ray crystallography studies of inhibitors complexed with Tsg101 protein are in progress to provide information for the design of higher affinity second-generation analogues by structure-based techniques. 3) Inhibitors of Proteosomal Targeting of Human Cytidine Deaminase APOBEC3G (A3G) Binding by the HIV-1 Protein Vif. After infection of a target cell and during reverse transcription of HIV-1 RNA into DNA, the human A3G enzyme deaminates C residues in the minus strand, converting them to U residues. This results in encoding of A residues in the plus strand DNA in place of the original G residues, with the net effect being hypermutation of proviral DNA. The HIV-1 23-kDa cytoplasmic protein Vif overcomes the antiviral effects of A3G by binding to it and targeting it for proteasomal degradation. The laboratory of Dr. Vinay Pathak (CCR, NCI) has shown that the Vif protein sequence S(144)LQYLA(149) is critical for targeting A3G to the proteosome. Our initial work has been to prepare fluoresceine isothiocyanate (FITC)-derived peptide conjugates for use in the laboratory of Dr. Robert Fisher (SAIC-Frederick, Inc.) to develop Vif fluorescence anisotropy (FA) binding assays. In collaboration with Dr. Pathak's laboratory, we will conduct structure-activity studies on the SLQYLA sequence to develop high-affinity, cell-permeable analogues that can effectively block proteosomal targeting of A3G by Vif. Among the techniques planned for this work is the use of hydrazone and oxime peptide libraries as described above for Tsg101-binding inhibitors.
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