A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

A novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model describing ceftazidime/avibactam efficacy against ß-lactamase-producing Gram-negative bacteria.

BACKGROUND: Diazabicyclooctanes (DBOs) are an increasingly important group of non ß-lactam ß-lactamase inhibitors, employed clinically in combinations such as ceftazidime/avibactam. The dose finding of such combinations is complicated using the traditional pharmacokinetic/pharmacodynamic (PK/PD) index approach, especially if the ß-lactamase inhibitor has an antibiotic effect of its own. OBJECTIVES: To develop a novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model for ceftazidime/avibactam against Gram-negative pathogens, with the potential for combination dosage simulation. METHODS: Four ß-lactamase-producing Enterobacteriaceae, covering Ambler classes A, B and D, were exposed to ceftazidime and avibactam, alone and in combination, in static time-kill experiments. A PKPD model was developed and evaluated using internal and external evaluation, and combined with a population PK model and applied in dosage simulations. RESULTS: The developed PKPD model included the effects of ceftazidime alone, avibactam alone and an 'enhancer' effect of avibactam on ceftazidime in addition to the ß-lactamase inhibitory effect of avibactam. The model could describe an extensive external Pseudomonas aeruginosa data set with minor modifications to the enhancer effect, and the utility of the model for clinical dosage simulation was demonstrated by investigating the influence of the addition of avibactam. CONCLUSIONS: A novel mechanism-based PKPD model for the DBO/ß-lactam combination ceftazidime/avibactam was developed that enables future comparison of the effect of avibactam with other DBO/ß-lactam inhibitors in simulations, and may be an aid in translating PKPD results from in vitro to animals and humans.

Nacubactam Enhances Meropenem Activity against Carbapenem-Resistant Klebsiella pneumoniae Producing KPC.

Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) ß-lactamase inhibitor that inactivates class A and C ß-lactamases and exhibits intrinsic antibiotic and ß-lactam "enhancer" activity against Enterobacteriaceae In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying blaKPC-2 or blaKPC-3; meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent Ki [Ki app] = 31 ± 3 µM) more efficiently than the K234R variant (Ki app = 270 ± 27 µM) and displayed a faster acylation rate (k2/K), which was 5,815 ± 582 M-1 s-1 for KPC-2 versus 247 ± 25 M-1 s-1 for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (+337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam's aminoethoxy tail formed unproductive interactions with the K234R variant's active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae Moreover, our data suggest that ß-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

Activity of nacubactam (RG6080/OP0595) combinations against MBL-producing Enterobacteriaceae.

BACKGROUND: Diazabicyclooctanes (DBOs) are promising ß-lactamase inhibitors. Some, including nacubactam (OP0595/RG6080), also bind PBP2 and have an enhancer effect, allowing activity against Enterobacteriaceae with MBLs, which DBOs do not inhibit. We tested the activity of nacubactam/ß-lactam combinations against MBL-producing Enterobacteriaceae. METHODS: Test panels comprised (i) 210 consecutive Enterobacteriaceae with NDM or VIM MBLs, as referred by UK diagnostic laboratories, and (ii) 99 supplementary MBL-producing Enterobacteriaceae, representing less prevalent phenotypes, species and enzymes. MICs were determined by CLSI agar dilution. RESULTS: MICs of nacubactam alone were bimodal, clustering at 1-8 mg/L or >32 mg/L; >85% of values for Escherichia coli and Enterobacter spp. fell into the low MIC cluster, whereas Proteeae were universally resistant and the Klebsiella spp. were divided between the two groups. Depending on the prospective breakpoint (4 + 4 or 8 + 4 mg/L), and on whether all isolates were considered or solely the Consecutive Collection, meropenem/nacubactam and cefepime/nacubactam inhibited 80.3%-93.3% of MBL producers, with substantial gains over nacubactam alone. Against the most resistant isolates (comprising 57 organisms with MICs of nacubactam >32 mg/L, cefepime ≥128 mg/L and meropenem ≥128 mg/L), cefepime/nacubactam at 8 + 4 mg/L inhibited 63.2% and meropenem/nacubactam at 8 + 4 mg/L inhibited 43.9%. Aztreonam/nacubactam, incorporating an MBL-stable ß-lactam partner, was almost universally active against the MBL producers and, unlike aztreonam/avibactam, had an enhancer effect. CONCLUSIONS: Nacubactam combinations, including those using MBL-labile ß-lactams, e.g. meropenem and cefepime, can overcome most MBL-mediated resistance. This behaviour reflects nacubactam's direct antibacterial and enhancer activity.

Escape mutations circumvent a tradeoff between resistance to a beta-lactam and resistance to a beta-lactamase inhibitor.

Beta-lactamase inhibitors are increasingly used to counteract antibiotic resistance mediated by beta-lactamase enzymes. These inhibitors compete with the beta-lactam antibiotic for the same binding site on the beta-lactamase, thus generating an evolutionary tradeoff: mutations that increase the enzyme's beta-lactamase activity tend to increase also its susceptibility to the inhibitor. Here, we investigate how common and accessible are mutants that escape this adaptive tradeoff. Screening a deep mutant library of the blaampC beta-lactamase gene of Escherichia coli, we identified mutations that allow growth at beta-lactam concentrations far exceeding those inhibiting growth of the wildtype strain, even in the presence of the enzyme inhibitor (avibactam). These escape mutations are rare and drug-specific, and some combinations of avibactam with beta-lactam drugs appear to prevent such escape phenotypes. Our results, showing differential adaptive potential of blaampC to combinations of avibactam and different beta-lactam antibiotics, suggest that it may be possible to identify treatments that are more resilient to evolution of resistance.

Efficacy of iclaprim against wild-type and thymidine kinase-deficient methicillin-resistant Staphylococcus aureus isolates in an in vitro fibrin clot model.

Iclaprim is a novel diaminopyrimidine antibiotic that is active against methicillin-resistant Staphylococcus aureus (MRSA). However, it is known that the activity of diaminopyrimidines against S. aureus is antagonized by thymidine through uptake and conversion to thymidylate by thymidine kinase. Unlike with humans, for whom thymidine levels are low, thymidine levels in rodents are high, thus precluding the accurate evaluation of iclaprim efficacy in animal models. We have studied the bactericidal activity of iclaprim against an isogenic pair of MRSA isolates, the wild-type parent AW6 and its thymidine kinase-deficient mutant AH1252, in an in vitro fibrin clot model. Clots, which were aimed at mimicking vegetation structure, were made from human or rat plasma containing either the parent AW6 or the mutant AH1252, and they were exposed to homologous serum supplemented with iclaprim (3.5 microg/ml), trimethoprim-sulfamethoxazole (TMP-SMX; 8/40 microg/ml), vancomycin (40 microg/ml), or saline, each of which was added one time for 48 h. In rat clots, iclaprim and TMP-SMX were bacteriostatic against the parent, AW6. In contrast, they were bactericidal (> or = 3 log10 CFU/clot killing of the original inoculum) against the mutant AH1252. Vancomycin was the most active drug against AW6 (P < 0.05), but it showed an activity similar those of iclaprim and TMP-SMX against AH1252. In human clots, iclaprim was bactericidal against both AW6 and AH1252 strains and was as effective as TMP-SMX and vancomycin (P > 0.05). Future studies of animals using simulated human kinetics of iclaprim and thymidine kinase-deficient MRSA, which eliminate the thymidine-induced confounding effect, are warranted to support the use of iclaprim in the treatment of severe MRSA infections in humans.

Increased hydrophobic interactions of iclaprim with Staphylococcus aureus dihydrofolate reductase are responsible for the increase in affinity and antibacterial activity.

OBJECTIVES: Iclaprim is a novel 2,4-diaminopyrimidine that exhibits potent, rapid bactericidal activity against major Gram-positive pathogens, including methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus, and is currently in clinical development for the treatment of complicated skin and skin structure infections. An understanding of the known mechanism of resistance to trimethoprim led to the design of this new inhibitor, with improved affinity towards dihydrofolate reductase (DHFR) from S. aureus and clinically useful activity against S. aureus including isolates resistant to trimethoprim. The objective of this study was to characterize the mode of action of iclaprim and its inhibitory properties against DHFR. METHODS: The mode of action of iclaprim was assessed by enzymatic analysis, direct binding studies, macromolecular synthesis profiles, synergy and antagonism studies to define its role as an inhibitor of DHFR. The binding properties of iclaprim to DHFR were compared with those of trimethoprim by X-ray crystallography. RESULTS: The enzymatic properties, direct binding and X-ray crystallographic studies delineated the mode of interaction with DHFR and the reason for the increased affinity of iclaprim towards the enzyme. The effect of iclaprim on bacterial physiology suggests that iclaprim behaves as a classical antibacterial DHFR inhibitor, as previously documented for trimethoprim. CONCLUSIONS: Iclaprim binds and inhibits bacterial DHFR in a similar manner to trimethoprim. However, the increased hydrophobic interactions between iclaprim and DHFR account for increased affinity and, unlike trimethoprim, enable iclaprim to inhibit even the resistant enzyme with nanomolar affinity, thus overcoming the mechanism of trimethoprim resistance. The increased antibacterial activity and lower propensity for resistance make iclaprim a clinically promising and useful inhibitor.

Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways.

An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.