Spiropyrimidinetriones: a Class of DNA Gyrase Inhibitors with Activity against Mycobacterium tuberculosis and without Cross-Resistance to Fluoroquinolones.

Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class operates via a novel mode of inhibition, which involves Mg2+-independent stabilization of the DNA cleavage complex with DNA gyrase and is thereby not cross-resistant with other DNA gyrase-inhibiting antibacterials, including fluoroquinolones. Compound 22 from the series was profiled broadly and showed in vitro cidality as well as intracellular activity against M. tuberculosis in macrophages. Evidence for the DNA gyrase mode of action was supported by inhibition of the target in a DNA supercoiling assay and elicitation of an SOS response seen in a recA reporter strain of M. tuberculosis. Pharmacokinetic properties of 22 supported evaluation of efficacy in an acute model of M. tuberculosis infection, where modest reduction in CFU numbers was seen. This work offers promise for deriving a novel drug class of tuberculosis agent without preexisting clinical resistance.

Untargeted Metabolomics To Ascertain Antibiotic Modes of Action.

Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5'-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanidem-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5'-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.

HIV capsid is a tractable target for small molecule therapeutic intervention.

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.

A low-molecular-weight entry inhibitor of both CCR5- and CXCR4-tropic strains of human immunodeficiency virus type 1 targets a novel site on gp41.

A low-molecular-weight human immunodeficiency virus type 1 (HIV-1) inhibitor, PF-68742 (molecular weight, 573), has been identified in a high-throughput screen for compounds that block HIV-1 envelope glycoprotein (Env)-mediated fusion. The compound is shown to be potent against R5 and X4 isolates in both cell-cell fusion and antiviral assays (50% effective concentrations of approximately 0.1 to 1 muM). Postfusion and HIV-1 pseudotyping control experiments confirm that PF-68742 is an entry inhibitor with Env as the specific target for antiviral action. PF-68742 was not able to block binding of monomeric gp120 to soluble CD4 or the binding of gp120:CD4 complexes to cell-associated CCR5, thus distinguishing PF-68742 from described gp120 antagonists and coreceptor binders. Escape variants of HIV-1(NL4-3) were selected, and all resistant viruses were found to contain a common G514R (HxB2 numbering) mutation in Env, located proximal to the furin cleavage site in the fusion peptide of gp41. When introduced into wild-type NL4-3 gp41, G514R conferred resistance to PF-68742. Resistance via G514R is shown to be associated with enhancement of virion infectivity by PF-68742 that may result from altered properties of inhibitor-bound Env, rather than from a loss of compound binding. Wild-type viruses and those with substitutions in the disulfide loop (DSL) region of gp41 were also examined for PF-68742 sensitivity. Here, complete resistance to PF-68742 was found to occur through changes outside of position 514, including in the gp41 DSL region. The results highlight PF-68742 as a starting point for novel therapies against HIV-1 and provide new insights into models of Env-mediated fusion.

Challenges and opportunities in the discovery, development, and commercialization of pathogen-targeted antibiotics.

The use of antibiotics directly correlates with the increase in antimicrobial resistance (AMR). Targeting novel antibiotics to patients with multidrug-resistant (MDR) pathogens should enhance their durability and slow development of resistance. The discovery, development, and clinical adoption of pathogen-targeted antibiotics have been hampered by technical and regulatory challenges. Growing insights into bacterial physiology and mechanisms of resistance, innovative clinical trial designs, streamlined regulatory approval pathways, and availability of rapid bacterial diagnostics are recent developments that can help address those challenges. Pathogen-targeted antibiotics provide an opportunity to treat patients with the right drug at the right time, leading to improved patient outcomes and better antimicrobial stewardship. Patient-centered pricing and reimbursement reform is needed to incentivize innovation.

Lersivirine, a nonnucleoside reverse transcriptase inhibitor with activity against drug-resistant human immunodeficiency virus type 1.

The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC(50)s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses.

New small-molecule inhibitor class targeting human immunodeficiency virus type 1 virion maturation.

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.

Pyrazole NNRTIs 1: design and initial optimisation of a novel template.

The design and synthesis of a novel series of non-nucleoside HIV reverse transcriptase inhibitors (NNRTIs) based on a pyrazole template is described. These compounds are active against wild type reverse transcriptase (RT) and retain activity against clinically important mutants.

Pyrazole NNRTIs 3: optimisation of physicochemical properties.

Our efforts to reduce overall lipophilicity and increase ligand-lipophilicity efficiency (LLE) by modification of the 3- and 5-substituents of pyrazole 1, a novel non-nucleoside HIV reverse transcriptase inhibitor (NNRTI) prototype were unsuccessful. In contrast replacement of the substituted benzyl group with corresponding phenylthio or phenoxy groups resulted in marked improvements in potency, ligand efficiency (LE) and LLE.

Pyrazole NNRTIs 4: selection of UK-453,061 (lersivirine) as a development candidate.

We prepared three discreet cohorts of potent non-nucleoside HIV reverse transcriptase inhibitors (NNRTIs) based on the recently reported 3-cyanophenoxypyrazole lead 3. Several of these compounds displayed very promising anti-HIV activity in vitro, safety, pharmacokinetic and pharmaceutical profiles. We describe our analysis and conclusions leading to the selection of alcohol 5 (UK-453,061, lersivirine) for clinical development.