The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CA NTD ) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CA NTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CA NTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CA NTD site 2) between CA helices 4, 5, and 7, on the surface of the CA NTD , that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid.

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

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The HIV-1 capsid (CA) protein, a domain of Gag, which participates in formation of both the mature and immature capsid, represents a potential target for anti-viral drug development. Characterization of hits obtained via high-throughput screening of an in vitro capsid assembly assay led to multiple compounds having this potential. We previously presented the characterization of two inhibitor series that bind the N-terminal domain of the capsid (CA(NTD)), at a site located at the bottom of its helical bundle, often referred to as the CAP-1 binding site. In this work we characterize a novel series of benzimidazole hits. Initial optimization of this series led to compounds with improved in vitro assembly and anti-viral activity. Using NMR spectroscopy we found that this series binds to a unique site on CA(NTD), located at the apex of the helical bundle, well removed from previously characterized binding sites for CA inhibitors. 2D (1)H-(15)N HSQC and (19)F NMR showed that binding of the benzimidazoles to this distinct site does not affect the binding of either cyclophilin A (CypA) to the CypA-binding loop or a benzodiazepine-based CA assembly inhibitor to the CAP-1 site. Unfortunately, while compounds of this series achieved promising in vitro assembly and anti-viral effects, they also were found to be quite sensitive to a number of naturally occurring CA(NTD) polymorphisms observed among clinical isolates. Despite the negative impact of this finding for drug development, the discovery of multiple inhibitor binding sites on CA(NTD) shows that capsid assembly is much more complex than previously realized.

Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3) isozymes and an APH(2؆) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3) and APH(2؆) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.Aminoglycosides are a class of commonly used broadspectrum antibiotics that target the bacterial ribosome. They are characterized by their signature chemical structure, an aminocyclitol nucleus. These antibiotics can be further categorized into two groups: the first group includes those that contain a 2-deoxystreptamine core, such as kanamycin, and the second, smaller group includes those that contain a non-2-deoxystreptamine core (Fig. 1A). Spectinomycin has a streptamine core and it is used in the treatment of acute gonococcal infections (1). Unfortunately, the efficacy of aminoglycosides has been compromised due to the continuous rise of drug resistance in pathogens. Resistance to aminoglycosides can be attributed to several mechanisms, of which enzymatic inactivation of the aminoglycoside is the most prevalent in the clinical setting. Aminoglycosides can be inactivated by the addition of an acetyl, a nucleotidyl, or a phosphate group by acetyltransferases, nucleotidyltransferases, or phosphotransferases (kinases; APHs), 5 respectively (2). Nomenclature of aminoglycoside-modifying enzymes follows the convention proposed by Shaw et al. (2): the type of modifying enzyme is identified by acetyltransferase, nucleotidyltransferase, or APH; this is followed by, in parentheses, the enzyme regiospecificity; then the substrate profile is designated by a roman numeral, and the unique amino acid sequence is denoted by a small letter.APHs generally yield high levels of resista...

Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with directacting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic. BI 201335, which is progressing through Phase IIb trials, contains a unique C-terminal carboxylic acid that binds noncovalently to the active site and a bromo-quinoline substitution on its proline residue that provides significant potency. In this work we have used stopped flow kinetics, x-ray crystallography, and NMR to characterize these distinctive features. Key findings include: slow association and dissociation rates within a singlestep binding mechanism; the critical involvement of water molecules in acid binding; and protein side chain rearrangements, a bromine-oxygen halogen bond, and profound pK a changes within the catalytic triad associated with binding of the bromoquinoline moiety.Worldwide, 130 -170 million people are chronically infected with hepatitis C virus (HCV) 2 (1). Infection can progress to cirrhosis, which can lead to hepatic decompensation or hepatocellular carcinoma. HCV infection can be cured by administering a combination of pegylated interferon and the nonspecific antiviral drug ribavirin for 24 -48 weeks. Unfortunately, this treatment is only 40 -50% successful in clearing genotype 1 infections, the most common type in most regions of the world (2). Clearly there is considerable need for more efficacious therapies.HCV is a small, positive-strand RNA virus with a 9600-nucleotide genome encoding a 3000-amino acid polyprotein that is subsequently processed into individual proteins by cellular and viral proteases. Four of the five cleavage sites between the NS (nonstructural) proteins are cleaved by the viral NS3 serine protease (EC 3.4.21.98). HCV NS3 is a bifunctional protein comprised of an N-terminal 180-amino acid serine protease domain and a C-terminal 420-amino acid helicase domain (3). The central portion of the 54-amino acid NS4A protein is structurally integrated into the NS3 protease domain (4) and is necessary for full protease activity (5).The search for specific, direct-acting, small molecule inhibitors of HCV replication began shortly after the virus was discovered in 1989 (6); the fact that no such drug has yet made it to the market attests to the challenging nature of this research. Throughout this period, the NS3-NS4A protease has remained a major focus of HCV drug discovery efforts. Beginning with the protease inhibitor BILN 2061 in 2002 (7, 8), a number of specific antivirals have been tested in human trials and have shown very promising results (9). The most advanced compounds in the clinic are NS3-NS4A protease inhibitors, with two, telaprevir and boceprevir, cu...