Ibrexafungerp: An orally active ß-1,3-glucan synthesis inhibitor.

We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious ß-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.

MK-5204: An orally active ß-1,3-glucan synthesis inhibitor.

Our previously reported efforts to produce an orally active ß-1,3-glucan synthesis inhibitor through the semi-synthetic modification of enfumafungin focused on replacing the C2 acetoxy moiety with an aminotetrazole and the C3 glycoside with a N,N-dimethylaminoether moiety. This work details further optimization of the C2 heterocyclic substituent, which identified 3-carboxamide-1,2,4-triazole as a replacement for the aminotetrazole with comparable antifungal activity. Alkylation of either the carboxamidetriazole at C2 or the aminoether at C3 failed to significantly improve oral efficacy. However, replacement of the isopropyl alpha amino substituent with a t-butyl, improved oral exposure while maintaining antifungal activity. These two structural modifications produced MK-5204, which demonstrated broad spectrum activity against Candida species and robust oral efficacy in a murine model of disseminated Candidiasis without the N-dealkylation liability observed for the previous lead.

In vitro studies evaluating the activity of imipenem in combination with relebactam against Pseudomonas aeruginosa.

BACKGROUND: The prevalence of antibiotic resistance is increasing, and multidrug-resistant Pseudomonas aeruginosa has been identified as a serious threat to human health. The production of ß-lactamase is a key mechanism contributing to imipenem resistance in P. aeruginosa. Relebactam is a novel ß-lactamase inhibitor, active against class A and C ß-lactamases, that has been shown to restore imipenem susceptibility. In a series of studies, we assessed the interaction of relebactam with key mechanisms involved in carbapenem resistance in P. aeruginosa and to what extent relebactam might overcome imipenem non-susceptibility. RESULTS: Relebactam demonstrated no intrinsic antibacterial activity against P. aeruginosa, had no inoculum effect, and was not subject to efflux. Enzymology studies showed relebactam is a potent (overall inhibition constant: 27 nM), practically irreversible inhibitor of P. aeruginosa AmpC. Among P. aeruginosa clinical isolates from the SMART global surveillance program (2009, n = 993; 2011, n = 1702; 2015, n = 5953; 2016, n = 6165), imipenem susceptibility rates were 68.4% in 2009, 67.4% in 2011, 70.4% in 2015, and 67.3% in 2016. With the addition of 4 µg/mL relebactam, imipenem susceptibility rates increased to 87.6, 86.0, 91.7, and 89.8%, respectively. When all imipenem-non-susceptible isolates were pooled, the addition of 4 µg/mL relebactam reduced the mode imipenem minimum inhibitory concentration (MIC) 8-fold (from 16 µg/mL to 2 µg/mL) among all imipenem-non-susceptible isolates. Of 3747 imipenem-non-susceptible isolates that underwent molecular profiling, 1200 (32%) remained non-susceptible to the combination imipenem/relebactam (IMI/REL); 42% of these encoded class B metallo-ß-lactamases, 11% encoded a class A GES enzyme, and no class D enzymes were detected. No relationship was observed between alleles of the chromosomally-encoded P. aeruginosa AmpC and IMI/REL MIC. CONCLUSIONS: IMI/REL exhibited potential in the treatment of carbapenem-resistant P. aeruginosa infections, with the exception of isolates encoding class B, some GES alleles, and class D carbapenemases.

Exploring the Pharmacokinetic/Pharmacodynamic Relationship of Relebactam (MK-7655) in Combination with Imipenem in a Hollow-Fiber Infection Model.

Resistance to antibiotics among bacterial pathogens is rapidly spreading, and therapeutic options against multidrug-resistant bacteria are limited. There is an urgent need for new drugs, especially those that can circumvent the broad array of resistance pathways that bacteria have evolved. In this study, we assessed the pharmacokinetic/pharmacodynamic relationship of the novel ß-lactamase inhibitor relebactam (REL; MK-7655) in a hollow-fiber infection model. REL is intended for use with the carbapenem ß-lactam antibiotic imipenem for the treatment of Gram-negative bacterial infections. In this study, we used an in vitro hollow-fiber infection model to confirm the efficacy of human exposures associated with the phase 2 doses (imipenem at 500 mg plus REL at 125 or 250 mg administered intravenously every 6 h as a 30-min infusion) against imipenem-resistant strains of Pseudomonas aeruginosa and Klebsiella pneumoniae Dose fractionation experiments confirmed that the pharmacokinetic parameter that best correlated with REL activity is the area under the concentration-time curve, consistent with findings in a murine pharmacokinetic/pharmacodynamic model. Determination of the pharmacokinetic/pharmacodynamic relationship between ß-lactam antibiotics and ß-lactamase inhibitors is complex, as there is an interdependence between their respective exposure-response relationships. Here, we show that this interdependence could be captured by treating the MIC of imipenem as dynamic: it changes with time, and this change is directly related to REL levels. For the strains tested, the percentage of the dosing interval time that the concentration remains above the dynamic MIC for imipenem was maintained at the carbapenem target of 30 to 40%, required for maximum efficacy, for imipenem at 500 mg plus REL at 250 mg.

Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography.

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a ß-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Broad coverage of genetically diverse strains of Clostridium difficile by actoxumab and bezlotoxumab predicted by in vitro neutralization and epitope modeling.

Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.

Novel orally active inhibitors of ß-1,3-glucan synthesis derived from enfumafungin.

The clinical success of the echinocandins, which can only be administered parentally, has validated ß-1,3-glucan synthase (GS) as an antifungal target. Semi-synthetic modification of enfumafungin, a triterpene glycoside natural product, was performed with the aim of producing a new class of orally active GS inhibitors. Replacement of the C2 acetoxy moiety with various heterocycles did not improve GS or antifungal potency. However, replacement of the C3 glycoside with an aminoether moiety dramatically improved oral pharmacokinetic (PK) properties while maintaining GS and antifungal potency. Installing an aminotetrazole at C2 in conjunction with an N-alkylated aminoether at C3 produced derivatives with significantly improved GS and antifungal potency that exhibited robust oral efficacy in a murine model of disseminated candidiasis.

Development of a fully automated platform for agar-based measurement of viable bacterial growth.

Dynamic in vitro antibacterial studies provide valuable insight on effective dosing strategies prior to translating to in vivo models. Frequent sampling is required to monitor the pharmacodynamics (PD) of these studies, leading to significant work when quantifying the bacterial load of the samples. Spreading a bacterial suspension on agar to allow colony counting is a proven process for measuring very low levels of growth, but commercial automation equipment to handle agar plating and colony counting at scale is not readily available. We describe a process to greatly decrease the hands-on time required for PD assays by utilizing general-purpose liquid handling robots to plate bacteria and a custom-made plate imager to automate colony counting. The platform developed handles the biological assay from beginning to end as well as sample tracking at each step of the process. The process relies heavily on custom automation scheduling software to enable dynamic process decisions and coordinate data flow throughout. Using the described platform, we can efficiently quantify >100 PD samples per day while maintaining the necessary dynamic range of the assay. Alleviating the main bottleneck in the dynamic antibacterial studies has allowed us to accelerate the rate of experiments to provide antibacterial dosing data within shorter timelines.

In Vitro Hollow-Fiber Studies Assessing Antibacterial Activity of Ceftolozane/Tazobactam Against Multidrug-Resistant Pseudomonas Aeruginosa.

Our hollow-fiber infection model simulated the projected steady-state pharmacokinetics of ceftolozane and tazobactam in lung epithelial lining fluid of patients with pneumonia receiving 3 g of ceftolozane/tazobactam every 8 hours. Results confirmed the previously established in vitro activity of ceftolozane/tazobactam at and above approved breakpoints against multidrug-resistant Pseudomonas aeruginosa, regardless of Pseudomonas-derived cephalosporinase allele.

A translational pharmacokinetic/pharmacodynamic model to characterize bacterial kill in the presence of imipenem-relebactam.

OBJECTIVES: Relebactam is a small molecule ß-lactamase inhibitor under clinical investigation for use as a fixed-dose combination with imipenem/cilastatin. Here we present a translational pharmacokinetic/pharmacodynamic mathematical model to support optimal dose selection of relebactam. METHODS: Data derived from in vitro checkerboard and hollow fiber infection studies of imipenem-resistant strains of Pseudomonas aeruginosa were incorporated into the model. The model integrates the effect of relebactam concentration on imipenem susceptibility in a semi-mechanistic manner using the checkerboard data and characterizes the bacterial time-kill profiles from the hollow fiber infection model data. RESULTS: Simulations demonstrated that the ratio of the area under the concentration-time curve for free drug to the minimum inhibitory concentration (fAUC/MIC) was the pharmacokinetic driver for relebactam, with a target fAUC/MIC=7.5 associated with 2-log kill. At a clinical dose of 250mg relebactam, greater than 2-log reductions in bacterial load are projected for imipenem-resistant strains with an imipenem/relebactam MIC≤4µg/mL. CONCLUSIONS: The study confirms that the pharmacokinetic/pharmacodynamic driver for relebactam is fAUC/MIC, that an fAUC/MIC ratio of 7.5 is associated with 2-log kill in vitro, and that a 250mg clinical dose of relebactam achieves this target value when delivered in combination with imipenem/cilastatin.

Development of an integrated semi-automated system for in vitro pharmacodynamic modelling.

OBJECTIVES: The aim of this study was to develop an integrated system for in vitro pharmacodynamic modelling of antimicrobials with greater flexibility, easier control and better accuracy than existing in vitro models. METHODS: Custom-made bottle caps, fittings, valve controllers and a modified bench-top shaking incubator were used. A temperature-controlled automated sample collector was built. Computer software was developed to manage experiments and to control the entire system including solenoid pinch valves, peristaltic pumps and the sample collector. The system was validated by pharmacokinetic simulations of linezolid 600 mg infusion. The antibacterial effect of linezolid against multiple Staphylococcus aureus strains was also studied in this system. RESULTS: An integrated semi-automated bench-top system was built and validated. The temperature-controlled automated sample collector allowed unattended collection and temporary storage of samples. The system software reduced the labour necessary for many tasks and also improved the timing accuracy for performing simultaneous actions in multiple parallel experiments. The system was able to simulate human pharmacokinetics of linezolid 600 mg intravenous infusion accurately. A pharmacodynamic study of linezolid against multiple S. aureus strains with a range of MICs showed that the required 24 h free drug AUC/MIC ratio was approximately 30 in order to keep the organism counts at the same level as their initial inoculum and was about > or = 68 in order to achieve > 2 log(10) cfu/mL reduction in the in vitro model. CONCLUSIONS: The integrated semi-automated bench-top system provided the ability to overcome many of the drawbacks of existing in vitro models. It can be used for various simple or complicated pharmacokinetic/pharmacodynamic studies efficiently and conveniently.

Epitopes and Mechanism of Action of the Clostridium difficile Toxin A-Neutralizing Antibody Actoxumab.

The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.

Elucidation of DnaE as the Antibacterial Target of the Natural Product, Nargenicin.

Resistance to existing classes of antibiotics drives the need for discovery of novel compounds with unique mechanisms of action. Nargenicin A1, a natural product with limited antibacterial spectrum, was rediscovered in a whole-cell antisense assay. Macromolecular labeling in both Staphylococcus aureus and an Escherichia coli tolC efflux mutant revealed selective inhibition of DNA replication not due to gyrase or topoisomerase IV inhibition. S. aureus nargenicin-resistant mutants were selected at a frequency of ∼1 × 10(-9), and whole-genome resequencing found a single base-pair change in the dnaE gene, a homolog of the E. coli holoenzyme α subunit. A DnaE single-enzyme assay was exquisitely sensitive to inhibition by nargenicin, and other in vitro characterization studies corroborated DnaE as the target. Medicinal chemistry efforts may expand the spectrum of this novel mechanism antibiotic.

Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties.

Emergence of bacterial resistance is a major issue for all classes of antibiotics; therefore, the identification of new classes is critically needed. Recently we reported the discovery of platensimycin by screening natural product extracts using a target-based whole-cell strategy with antisense silencing technology in concert with cell free biochemical validations. Continued screening efforts led to the discovery of platencin, a novel natural product that is chemically and biologically related but different from platensimycin. Platencin exhibits a broad-spectrum Gram-positive antibacterial activity through inhibition of fatty acid biosynthesis. It does not exhibit cross-resistance to key antibiotic resistant strains tested, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, and vancomycin-resistant Enterococci. Platencin shows potent in vivo efficacy without any observed toxicity. It targets two essential proteins, beta-ketoacyl-[acyl carrier protein (ACP)] synthase II (FabF) and III (FabH) with IC50 values of 1.95 and 3.91 microg/ml, respectively, whereas platensimycin targets only FabF (IC50 = 0.13 microg/ml) in S. aureus, emphasizing the fact that more antibiotics with novel structures and new modes of action can be discovered by using this antisense differential sensitivity whole-cell screening paradigm.

Caspofungin susceptibility in Aspergillus and non-Aspergillus molds: inhibition of glucan synthase and reduction of beta-D-1,3 glucan levels in culture.

Caspofungin inhibits synthesis of beta-D-1,3 glucan, essential to cell walls in Candida and Aspergillus spp., but activity against less common molds is largely uncharacterized. We demonstrate that caspofungin inhibits beta-D-1,3 glucan synthesis and reduces in vitro growth of clinical isolates from the genera Alternaria, Curvularia, Scedosporium, Acremonium, Bipolaris, and Trichoderma.