The diaryl ethers are a novel class of antituberculosis drug candidates that inhibit InhA, the enoyl-ACP reductase involved in the fatty acid biosynthesis (FASII) pathway, and have antibacterial activity against both drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis. In the present work we demonstrate that two time-dependent B-ring modified diaryl ether InhA inhibitors have antibacterial activity in a mouse model of TB infection when delivered by intraperitoneal injection. We propose that the efficacy of these compounds is related to their residence time on the enzyme, and to identify structural features that modulate drug-target residence time in this system, we have explored the inhibition of InhA by a series of B-ring modified analogues. Seven ortho substituted compounds were found to be time dependent inhibitors of InhA where the slow step leading to the final EI* complex is thought to correlate with closure and ordering of the InhA substrate binding loop. A detailed mechanistic understanding of the molecular basis for residence time in this system will facilitate the development of InhA inhibitors with improved in vivo activity.

We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-Inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-Inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 M) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-Inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand-protein interactions.H IV-1 encodes three enzymes that are essential for virus replication: reverse transcriptase, protease, and integrase (IN). To date, only the first two proteins have been exploited as therapeutic targets. The continuing emergence of new HIV-1 variants resistant to current treatments, together with cytotoxicity problems, makes the search for new anti-HIV-1 drugs imperative. HIV-1 IN is an attractive antiviral drug target that has no known human counterparts. Furthermore, the use of a common active site for 3Ј end processing and DNA strand transfer constrain the range of mutations that can contribute to drug resistance without compromising catalytic activity.HIV-1 IN catalyzes integration of the viral DNA, made by reverse transcription, into the host chromosome in a two-step reaction (reviewed in ref. 1). In the first step, called 3Ј processing, two nucleotides are removed at each 3Ј end of the viral DNA. In the next step, called DNA strand transfer, concerted transesterification reactions integrate the viral DNA ends into the host genome. In vivo, the enzyme acts in the context of a large nucleoprotein complex termed the ''preintegration complex'' (PIC). A number of viral proteins and host factors assemble within the PIC to orchestrate the integration process (2-10).HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (residues 1-50), which contains the HHCC zinc-binding motif; the core domain (residues 51-212), which contains the catalytic site; and the Cterminal domain (residues 213-270), which is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (11-15). In addition,...

HIV-1 integrase binds site-specifically to the ends of the viral cDNA. We used two HIV-1 integrase-DNA cross-linking assays to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivities. The disulfide assay probes cross-linking between the integrase residue 148 and the 5Ј-terminal cytosine of the viral cDNA, and the Schiff base assay probes cross-linking between an integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Cross-linking interference by eight integrase inhibitors shows that the most potent cross-linking inhibitors are 3Ј-processing inhibitors, indicating that crosslinking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak cross-linking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific integrase-inhibitor contacts that prevent cross-linking formation. Four inhibitors that prevented Schiff base cross-linking to the conserved 3Ј-terminal adenine position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand cross-linking, whereas the other two had a more global effect on integrase-DNA binding. These findings have implications for elucidating inhibitor binding sites and mechanisms of action. The cross-linking assays also provide clues to the molecular interactions between integrase and the viral cDNA.

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Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3-processing (3-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3-processing site. 3-Processing was blocked with substrates containing constrained sugars and ␣-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3-P site inhibited 3-P, suggesting the importance of the minor groove contacts for 3-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Positionspecific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors. HIV-12 integrase (integrase) catalyzes insertion of cDNA copies of the viral genome into human chromosomes. Integrase binds to the ends ("att" sites) of each viral long terminal repeat (LTR) through sequence-specific recognition of a conserved 5Ј-CA within the sequence 5Ј-GCAGT. In the first of two reactions, integrase cleaves the 3Ј-ends of the viral DNA, releasing the terminal 5Ј-GT dinucleotide (3Ј-processing, 3Ј-P). In the second reaction, the free 3Ј-hydroxyl of the conserved adenine provides the nucleophile for insertion of the viral cDNA into a chromosome (strand transfer, ST). Gap repair and ligation between the viral and cellular DNA are performed by cellular factors. (For recent reviews and insights on integration, see Refs. 1-5.)Determination of the molecular interactions between integrase and its DNA substrates (viral and chromosomal DNA) has proven challenging, and a co-crystal of these components remains elusive. Biochemical studies have revealed contact points between the viral DNA and integrase. Integrase has an absolute requirement for the conserved 5Ј-CA adjoining the 3Ј-P site (underlined in Fig. 1A). The efficiency of 3Ј-P is also dramatically decreased by changes to the G immediately 5Ј to the conserved CA dinucleotide (6 -8). The conserved adenine, substituted by 5-iododeoxyuracil as a photocross-linker, forms a photocross-link to Lys-159 of integrase (9). Residue Lys-159 also contacts the phosphate 5Ј to the conserved deoxyadenosine (10). Mutagenesis showed that Tyr-143 and probably Gln-148 interact with the 5Ј-overhang resulting from 3Ј-P (9, 11). Moreover, disulfide cross-linking ...

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