FDA, CDC, and NIH Co-sponsored Public Workshop Summary-Development Considerations of Antimicrobial Drugs for the Treatment of Gonorrhea.

There is an unmet need for developing drugs for the treatment of gonorrhea, due to rapidly evolving resistance of Neisseria gonorrhoeae against antimicrobial drugs used for empiric therapy, an increase in globally reported multidrug resistant cases, and the limited available therapeutic options. Furthermore, few drugs are under development. Development of antimicrobials is hampered by challenges in clinical trial design, limitations of available diagnostics, changes in and varying standards of care, lack of robust animal models, and clinically relevant pharmacodynamic targets. On April 23, 2021, the U.S. Food and Drug Administration; Centers for Disease Control and Prevention; and National Institute of Allergy and Infectious Diseases, National Institutes of Health co-sponsored a workshop with stakeholders from academia, industry, and regulatory agencies to discuss the challenges and strategies, including potential collaborations and incentives, to facilitate the development of drugs for the treatment of gonorrhea. This article provides a summary of the workshop.

Evaluation of a panel of therapeutic antibody clinical candidates for efficacy against SARS-CoV-2 in Syrian hamsters.

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (-24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.

SARS-CoV-2 BA.1 variant is neutralized by vaccine booster-elicited serum but evades most convalescent serum and therapeutic antibodies.

The rapid spread of the highly contagious Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) along with its high number of mutations in the spike gene has raised alarms about the effectiveness of current medical countermeasures. To address this concern, we measured the neutralization of the Omicron BA.1 variant pseudovirus by postvaccination serum samples after two and three immunizations with the Pfizer/BioNTech162b2 SARS-CoV-2 mRNA (Pfizer/BNT162b2) vaccine, convalescent serum samples from unvaccinated individuals infected by different variants, and clinical-stage therapeutic antibodies. We found that titers against the Omicron variant were low or undetectable after two immunizations and in many convalescent serum samples, regardless of the infecting variant. A booster vaccination increased titers more than 30-fold against Omicron to values comparable to those seen against the D614G variant after two immunizations. Neither age nor sex was associated with the differences in postvaccination antibody responses. We also evaluated 18 clinical-stage therapeutic antibody products and an antibody mimetic protein product obtained directly from the manufacturers. Five monoclonal antibodies, the antibody mimetic protein, three antibody cocktails, and two polyclonal antibody preparations retained measurable neutralization activity against Omicron with a varying degree of potency. Of these, only three retained potencies comparable to the D614G variant. Two therapeutic antibody cocktails in the tested panel that are authorized for emergency use in the United States did not neutralize Omicron. These findings underscore the potential benefit of mRNA vaccine boosters for protection against Omicron and the need for rapid development of antibody therapeutics that maintain potency against emerging variants.

Key Substitutions in the Spike Protein of SARS-CoV-2 Variants Can Predict Resistance to Monoclonal Antibodies, but Other Substitutions Can Modify the Effects.

Mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.429 (epsilon), B.1.526 (iota), A.23.1, and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies. IMPORTANCE Therapeutic antibodies are effective in preventing severe disease from SARS-CoV-2 infection (COVID-19), but their effectiveness may be reduced by virus variants with mutations affecting the spike protein. To help predict resistance to therapeutic antibodies in emerging variants, we profiled resistance patterns of 25 antibody products in late stages of clinical development against a large panel of variants that include single and multiple substitutions found in the spike protein. We found that the presence of a key substitution in variants with multiple spike substitutions can predict resistance against a variant but that other substitutions can affect the degree of resistance in unpredictable ways. These findings highlight complex interactions among substitutions in the spike protein affecting virus neutralization and, potentially, virus entry into cells.

SARS-CoV-2 Delta Variant Displays Moderate Resistance to Neutralizing Antibodies and Spike Protein Properties of Higher Soluble ACE2 Sensitivity, Enhanced Cleavage and Fusogenic Activity.

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants' spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

SARS-COV-2 Delta variant displays moderate resistance to neutralizing antibodies and spike protein properties of higher soluble ACE2 sensitivity, enhanced cleavage and fusogenic activity.

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. The spike proteins of B.1.617.1, B.1.617.2, and AY.1 variants have several substitutions in the receptor binding domain (RBD), including L452R+E484Q, L452R+T478K, and K417N+L452R+T478K, respectively, that could potentially reduce effectiveness of therapeutic antibodies and current vaccines. Here we compared B.1.617 variants, and their single and double RBD substitutions for resistance to neutralization by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. Pseudoviruses with the B.1.617.1, B.1.617.2, and AY.1 spike showed a modest 1.5 to 4.4-fold reduction in neutralization titer by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for pseudoviruses with C.37, P.1, R.1, and B.1.526 spikes, but seven- and sixteen-fold reduction for vaccine-elicited and convalescent sera, respectively, was seen for pseudoviruses with the B.1.351 spike. Four of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses due to the L452R substitution, whereas six of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.1 pseudoviruses due to either the E484Q or L452R substitution. Against AY.1 pseudoviruses, the L452R and K417N substitutions accounted for the loss of neutralization by four antibodies and one antibody, respectively, whereas one antibody lost potency that could not be fully accounted for by a single RBD substitution. The modest resistance of B.1.617 variants to vaccine-elicited sera suggest that current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants, but the therapeutic antibodies need to be carefully selected based on their resistance profiles. Finally, the spike proteins of B.1.617 variants are more efficiently cleaved due to the P681R substitution, and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster.

The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants. ONE SENTENCE SUMMARY: Third dose of Pfizer/BioNTech COVID-19 vaccine significantly boosts neutralizing antibodies to the Omicron variant compared to a second dose, while neutralization of Omicron by convalescent sera, two-dose vaccine-elicited sera, or therapeutic antibodies is variable and often low.

Determining the optimal dosing of a novel combination regimen of ceftazidime/avibactam with aztreonam against NDM-1-producing Enterobacteriaceae using a hollow-fibre infection model.

BACKGROUND: MBL-producing strains of Enterobacteriaceae are a major public health concern. We sought to define optimal combination regimens of ceftazidime/avibactam with aztreonam in a hollow-fibre infection model (HFIM) of MBL-producing strains of Escherichia coli and Klebsiella pneumoniae. METHODS: E. coli ARLG-1013 (blaNDM-1, blaCTX-M, blaCMY, blaTEM) and K. pneumoniae ARLG-1002 (blaNDM-1, blaCTXM-15, blaDHA, blaSHV, blaTEM) were studied in the HFIM using simulated human dosing regimens of ceftazidime/avibactam and aztreonam. Experiments were designed to evaluate the effect of staggered versus simultaneous administration, infusion duration and aztreonam daily dose (6 g/day versus 8 g/day) on bacterial killing and resistance suppression. Prospective validation experiments for the most active combination regimens were performed in triplicate to ensure reproducibility. RESULTS: Staggered administration of the combination (ceftazidime/avibactam followed by aztreonam) was found to be inferior to simultaneous administration. Longer infusion durations (2 h and continuous infusion) also resulted in enhanced bacterial killing relative to 30 min infusions. The rate of killing was more pronounced with 8 g/day versus 6 g/day aztreonam combination regimens for both tested strains. In the prospective validation experiments, ceftazidime/avibactam with aztreonam dosed every 8 and 6 h, respectively (ceftazidime/avibactam 2/0.5 g every 8 h + aztreonam 2 g every 6 h), or ceftazidime/avibactam with aztreonam as continuous infusions resulted in maximal bacterial killing and resistance suppression over 7 days. CONCLUSIONS: Simultaneous administration of aztreonam 8 g/day given as a continuous or 2 h infusion with ceftazidime/avibactam resulted in complete bacterial eradication and resistance suppression. Further study of this combination is needed with additional MBL-producing Gram-negative pathogens. The safety of this double ß-lactam strategy also warrants further study in Phase 1 clinical trials.

In Vivo Activity of Amodiaquine against Ebola Virus Infection.

During the Ebola virus disease (EVD) epidemic in Western Africa (2013‒2016), antimalarial treatment was administered to EVD patients due to the high coexisting malaria burden in accordance with World Health Organization guidelines. In an Ebola treatment center in Liberia, EVD patients receiving the combination antimalarial artesunate-amodiaquine had a lower risk of death compared to those treated with artemether-lumefantrine. As artemether and artesunate are derivatives of artemisinin, the beneficial anti-Ebola virus (EBOV) effect observed could possibly be attributed to the change from lumefantrine to amodiaquine. Amodiaquine is a widely used antimalarial in the countries that experience outbreaks of EVD and, therefore, holds promise as an approved drug that could be repurposed for treating EBOV infections. We investigated the potential anti-EBOV effect of amodiaquine in a well-characterized nonhuman primate model of EVD. Using a similar 3-day antimalarial dosing strategy as for human patients, plasma concentrations of amodiaquine in healthy animals were similar to those found in humans. However, the treatment regimen did not result in a survival benefit or decrease of disease signs in EBOV-infected animals. While amodiaquine on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of amodiaquine when used in combination with artesunate or another antiviral.

Generating Robust and Informative Nonclinical In Vitro and In Vivo Bacterial Infection Model Efficacy Data To Support Translation to Humans.

In June 2017, the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, organized a workshop entitled "Pharmacokinetics-Pharmacodynamics (PK/PD) for Development of Therapeutics against Bacterial Pathogens." The aims were to discuss details of various PK/PD models and identify sound practices for deriving and utilizing PK/PD relationships to design optimal dosage regimens for patients. Workshop participants encompassed individuals from academia, industry, and government, including the United States Food and Drug Administration. This and the accompanying review on clinical PK/PD summarize the workshop discussions and recommendations. Nonclinical PK/PD models play a critical role in designing human dosage regimens and are essential tools for drug development. These include in vitro and in vivo efficacy models that provide valuable and complementary information for dose selection and translation from the laboratory to human. It is crucial that studies be designed, conducted, and interpreted appropriately. For antibacterial PK/PD, extensive published data and expertise are available. These have been leveraged to develop recommendations, identify common pitfalls, and describe the applications, strengths, and limitations of various nonclinical infection models and translational approaches. Despite these robust tools and published guidance, characterizing nonclinical PK/PD relationships may not be straightforward, especially for a new drug or new class. Antimicrobial PK/PD is an evolving discipline that needs to adapt to future research and development needs. Open communication between academia, pharmaceutical industry, government, and regulatory bodies is essential to share perspectives and collectively solve future challenges.

Pharmacokinetic Data Are Predictive of In Vivo Efficacy for Cefixime and Ceftriaxone against Susceptible and Resistant Neisseria gonorrhoeae Strains in the Gonorrhea Mouse Model.

There is a pressing need for drug development for gonorrhea. Here we describe a pharmacokinetic (PK)/pharmacodynamic (PD) analysis of extended-spectrum cephalosporins (ESC) against drug-susceptible and drug-resistant gonococcal strains in a murine genital tract infection model. The PK determined in uninfected mice displayed a clear dose-response in plasma levels following single doses of ceftriaxone (CRO) (intraperitoneal) or cefixime (CFM) (oral). The observed doses required for efficacy against ESC-susceptible (ESCs) strain FA1090 were 5 mg/kg of body weight (CRO) and 12 mg/kg (CFM); these doses had estimated therapeutic times (the time that the free drug concentration remains above the MIC [fTMIC]) of 24 h and 37 h, respectively. No single dose of CRO or CFM was effective against ESC-resistant (ESCr) strain H041. However, fractionation (three times a day every 8 h [TIDq8h]) of a 120-mg/kg dose of CRO resulted in estimated therapeutic times in the range of 23 h and cleared H041 infection in a majority (90%) of mice, comparable to the findings for gentamicin. In contrast, multiple CFM doses of 120 or 300 mg/kg administered TIDq8h cleared infection in ≤50% of mice, with the therapeutic times estimated from single-dose PK data being 13 and 27 h, respectively. This study reveals a clear relationship between plasma ESC levels and bacterial clearance rates in the gonorrhea mouse model. The PK/PD relationships observed in mice reflected those observed in humans, with in vivo efficacy against an ESCs strain requiring doses that yielded an fTMIC in excess of 20 to 24 h. PK data also accurately predicted the failure of single doses of ESCs against an ESCr strain and were useful in designing effective dosing regimens.

Evaluation of the Activity of Lamivudine and Zidovudine against Ebola Virus.

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 µM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 µg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.

Pharmacodynamic Profiling of a Siderophore-Conjugated Monocarbam in Pseudomonas aeruginosa: Assessing the Risk for Resistance and Attenuated Efficacy.

The objective of this study was to investigate the risk of attenuated efficacy due to adaptive resistance for the siderophore-conjugated monocarbam SMC-3176 in Pseudomonas aeruginosa by using a pharmacokinetic/pharmacodynamic (PK/PD) approach. MICs were determined in cation-adjusted Mueller-Hinton broth (MHB) and in Chelex-treated, dialyzed MHB (CDMHB). Spontaneous resistance was assessed at 2× to 16× the MIC and the resulting mutants sequenced. Efficacy was evaluated in a neutropenic mouse thigh model at 3.13 to 400 mg/kg of body weight every 3 h for 24 h and analyzed for association with free time above the MIC (fT>MIC). To closer emulate the conditions of the in vivo model, we developed a novel assay testing activity mouse whole blood (WB). All mutations were found in genes related to iron uptake: piuA, piuC, pirR, fecI, and pvdS. Against four P. aeruginosa isolates, SMC-3176 displayed predictable efficacy corresponding to the fT>MIC using the MIC in CDMHB (R(2) = 0.968 to 0.985), with stasis to 2-log kill achieved at 59.4 to 81.1%. Efficacy did not translate for P. aeruginosa isolate JJ 4-36, as the in vivo responses were inconsistent with fT>MIC exposures and implied a threshold concentration that was greater than the MIC. The results of the mouse WB assay indicated that efficacy was not predictable using the MIC for JJ 4-36 and four additional isolates, against which in vivo failures of another siderophore-conjugated ß-lactam were previously reported. SMC-3176 carries a risk of attenuated efficacy in P. aeruginosa due to rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems. Substantial in vivo testing is warranted for compounds using the siderophore approach to thoroughly screen for this in vitro-in vivo disconnect in P. aeruginosa.

Pharmacokinetics/pharmacodynamics of a ß-lactam and ß-lactamase inhibitor combination: a novel approach for aztreonam/avibactam.

OBJECTIVES: The combination of aztreonam/avibactam has promising activity against MDR Gram-negative pathogens producing metallo-ß-lactamases (MBLs), such as New Delhi MBL-1. Pharmacokinetic (PK)/pharmacodynamic (PD) understanding of this combination is critical for optimal clinical dose selection. This study focuses on the determination of an integrated PK/PD approach for aztreonam/avibactam across multiple clinical Enterobacteriaceae strains. METHODS: Six clinical Enterobacteriaceae isolates expressing MBLs and ESBLs were studied in an in vitro hollow-fibre infection model (HFIM) using various dosing regimens simulating human-like PK for aztreonam/avibactam. The neutropenic murine thigh infection model was used for in vivo validation against two bacterial strains. RESULTS: MIC values of aztreonam/avibactam for the isolates ranged from 0.125 to 8 mg/L. Using a constant infusion of avibactam at 4 mg/L, the aztreonam PK/PD index was observed as % fT >MIC. Studies performed in the presence of a fixed dose of aztreonam revealed that the efficacy of avibactam correlates best with percentage of time above a critical threshold concentration of 2-2.5 mg/L. These conclusions translated well to the efficacy observed in the murine thigh model, demonstrating in vivo validation of the in vitro PK/PD target. CONCLUSIONS: PK/PD evaluations for aztreonam/avibactam in HFIM yielded a single target across strains with a wide MIC range. This integrated approach could be easily applied for forecasting clinically efficacious doses for ß-lactam/ß-lactamase inhibitor combinations.

Optimization of pyrrolamide topoisomerase II inhibitors toward identification of an antibacterial clinical candidate (AZD5099).

AZD5099 (compound 63) is an antibacterial agent that entered phase 1 clinical trials targeting infections caused by Gram-positive and fastidious Gram-negative bacteria. It was derived from previously reported pyrrolamide antibacterials and a fragment-based approach targeting the ATP binding site of bacterial type II topoisomerases. The program described herein varied a 3-piperidine substituent and incorporated 4-thiazole substituents that form a seven-membered ring intramolecular hydrogen bond with a 5-position carboxylic acid. Improved antibacterial activity and lower in vivo clearances were achieved. The lower clearances were attributed, in part, to reduced recognition by the multidrug resistant transporter Mrp2. Compound 63 showed notable efficacy in a mouse neutropenic Staphylococcus aureus infection model. Resistance frequency versus the drug was low, and reports of clinical resistance due to alteration of the target are few. Hence, 63 could offer a novel treatment for serious issues of resistance to currently used antibacterials.

Fragment-to-hit-to-lead discovery of a novel pyridylurea scaffold of ATP competitive dual targeting type II topoisomerase inhibiting antibacterial agents.

The discovery and optimization of a new class of bacterial topoisomerase (DNA gyrase and topoisomerase IV) inhibitors binding in the ATP domain are described. A fragment molecule, 1-ethyl-3-(2-pyridyl)urea, provided sufficiently potent enzyme inhibition (32 µM) to prompt further analogue work. Acids and acid isosteres were incorporated at the 5-pyridyl position of this fragment, bridging to a key asparagine residue, improving enzyme inhibition, and leading to measurable antibacterial activity. A CF3-thiazole substituent at the 4-pyridyl position improved inhibitory potency due to a favorable lipophilic interaction. Promising antibacterial activity was seen versus the Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae and the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis . Precursor metabolite incorporation and mutant analysis studies support the mode-of-action, blockage of DNA synthesis by dual target topoisomerase inhibition. Compound 35 was efficacious in a mouse S. aureus disease model, where a 4.5-log reduction in colony forming units versus control was demonstrated.

Pyrrolamide DNA gyrase inhibitors: fragment-based nuclear magnetic resonance screening to identify antibacterial agents.

DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 µM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.

Pyrrolamide DNA gyrase inhibitors: optimization of antibacterial activity and efficacy.

The pyrrolamides are a new class of antibacterial agents targeting DNA gyrase, an essential enzyme across bacterial species and inhibition results in the disruption of DNA synthesis and subsequently, cell death. The optimization of biochemical activity and other drug-like properties through substitutions to the pyrrole, piperidine, and heterocycle portions of the molecule resulted in pyrrolamides with improved cellular activity and in vivo efficacy.

A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase.

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo.

DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.