Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.

Molecular drivers of resistance to sulbactam-durlobactam in contemporary clinical isolates of Acinetobacter baumannii.

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe infections that are difficult to treat due to pre-existing antibiotic resistance. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat serious infections caused by Acinetobacter, including multidrug- and carbapenem-resistant strains. In a recent global surveillance study of 5,032 ABC clinical isolates collected from 2016 to 2021, less than 2% of ABC isolates had SUL-DUR MIC values >4 µg/mL. Molecular characterization of these isolates confirmed the primary drivers of resistance are metallo-ß-lactamases or penicillin-binding protein 3 (PBP3) mutations, as previously described. In addition, this study shows that certain common PBP3 variants, such as A515V, are insufficient to confer sulbactam resistance and that the efflux of durlobactam by AdeIJK is likely to play a role in a subset of strains.

Bacterial Cytological Profiling Identifies Rhodanine-Containing PAINS Analogs as Specific Inhibitors of Escherichia coli Thymidylate Kinase In Vivo.

In this study, we sought to determine whether an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

In Vitro Antibacterial Activity and In Vivo Efficacy of Sulbactam-Durlobactam against Pathogenic Burkholderia Species.

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, Burkholderia mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen ß-lactamase, which confers resistance to ß-lactams. The ß-lactam-ß-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. In this study, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured MICs of SUL-DUR against BCC and B. gladioli (n = 150), B. mallei (n = 30), and B. pseudomallei (n = 28), studied the kinetics of inhibition of the PenA1 ß-lactamase from B. multivorans and the PenI ß-lactamase from B. pseudomallei by durlobactam, tested for blaPenA1 induction by SUL-DUR, and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 µg/ml. Durlobactam potently inhibited PenA1 and PenI with second-order rate constant for inactivation (k2/K) values of 3.9 × 106 M-1 s-1 and 2.6 × 103 M-1 s-1 and apparent Ki (Kiapp) of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest that SUL-DUR may be useful as a treatment for Burkholderia infections.

N-Hydroxyformamide LpxC inhibitors, their in vivo efficacy in a mouse Escherichia coli infection model, and their safety in a rat hemodynamic assay.

UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC), the zinc metalloenzyme catalyzing the first committed step of lipid A biosynthesis in Gram-negative bacteria, has been a target for antibacterial drug discovery for many years. All inhibitor chemotypes reaching an advanced preclinical stage and clinical phase 1 have contained terminal hydroxamic acid, and none have been successfully advanced due, in part, to safety concerns, including hemodynamic effects. We hypothesized that the safety of LpxC inhibitors could be improved by replacing the terminal hydroxamic acid with a different zinc-binding group. After choosing an N-hydroxyformamide zinc-binding group, we investigated the structure-activity relationship of each part of the inhibitor scaffold with respect to Pseudomonas aeruginosa and Escherichia coli LpxC binding affinity, in vitro antibacterial potency and pharmacological properties. We identified a novel, potency-enhancing hydrophobic binding interaction for an LpxC inhibitor. We demonstrated in vivo efficacy of one compound in a neutropenic mouse E. coli infection model. Another compound was tested in a rat hemodynamic assay and was found to have a hypotensive effect. This result demonstrated that replacing the terminal hydroxamic acid with a different zinc-binding group was insufficient to avoid this previously recognized safety issue with LpxC inhibitors.

Frequency and Mechanism of Spontaneous Resistance to Sulbactam Combined with the Novel ß-Lactamase Inhibitor ETX2514 in Clinical Isolates of Acinetobacter baumannii.

The novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine ß-lactamase inhibitor that restores sulbactam activity against resistant Acinetobacter baumannii The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10-10 to <9.0 × 10-10 at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to ß-lactamase inhibition, ETX2514 may enhance sulbactam activity in A. baumannii via inhibition of PBP2.

Kinetics of Sulbactam Hydrolysis by ß-Lactamases, and Kinetics of ß-Lactamase Inhibition by Sulbactam.

Sulbactam is one of four ß-lactamase inhibitors in current clinical use to counteract drug resistance caused by degradation of ß-lactam antibiotics by these bacterial enzymes. As a ß-lactam itself, sulbactam is susceptible to degradation by ß-lactamases. I investigated the Michaelis-Menten kinetics of sulbactam hydrolysis by 14 ß-lactamases, representing clinically widespread groups within all four Ambler classes, i.e., CTX-M-15, KPC-2, SHV-5, and TEM-1 for class A; IMP-1, NDM-1, and VIM-1 for class B; Acinetobacter baumannii ADC-7, Pseudomonas aeruginosa AmpC, and Enterobacter cloacae P99 for class C; and OXA-10, OXA-23, OXA-24, and OXA-48 for class D. All of the ß-lactamases were able to hydrolyze sulbactam, although they varied widely in their kinetic constants for the reaction, even within each class. I also investigated the inactivation kinetics of the inhibition of these enzymes by sulbactam. The class A ß-lactamases varied widely in their susceptibility to inhibition, the class C and D enzymes were very weakly inhibited, and the class B enzymes were essentially or completely unaffected. In addition, we measured the sulbactam turnover number, the sulbactam/enzyme molar ratio required for complete inhibition of each enzyme. Class C enzymes had the lowest turnover numbers, class A enzymes varied widely, and class D enzymes had very high turnover numbers. These results are valuable for understanding which ß-lactamases ought to be well inhibited by sulbactam. Moreover, since sulbactam has intrinsic antibacterial activity against Acinetobacter species pathogens, these results contribute to understanding ß-lactamase-mediated sulbactam resistance in Acinetobacter, especially due to the action of the widespread class D enzymes.

Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914.

We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and ß.

The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability.

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

Target-based resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a novel bacterial type II topoisomerase inhibitor.

In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

Molecular mechanisms of sulbactam antibacterial activity and resistance determinants in Acinetobacter baumannii.

Sulbactam is a class A ß-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.

Nuclear Targeting with an Auger Electron Emitter Potentiates the Action of a Widely Used Antineoplastic Drug.

We present the combination of the clinically well-proven chemotherapeutic agent, Doxorubicin, and (99m)Tc, an Auger and internal conversion electron emitter, into a dual-action agent for therapy. Chemical conjugation of Doxorubicin to (99m)Tc afforded a construct which autonomously ferries a radioactive payload into the cell nucleus. At this site, damage is exerted by dose deposition from Auger radiation. The (99m)Tc-conjugate exhibited a dose-dependent inhibition of survival in a selected panel of cancer cells and an in vivo study in healthy mice evidenced a biodistribution which is comparable to that of the parent drug. The homologous Rhenium conjugate was found to effectively bind to DNA, inhibited human Topoisomerase II, and exhibited cytotoxicity in vitro. The collective in vitro and in vivo data demonstrate that the presented metallo-conjugates closely mimic native Doxorubicin.

Novel broad-spectrum inhibitors of bacterial methionine aminopeptidase.

With increasing emergence of multi-drug resistant infections, there is a dire need for new classes of compounds that act through unique mechanisms. In this work, we describe the discovery and optimization of a novel series of inhibitors of bacterial methionine aminopeptidase (MAP). Through a high-throughput screening campaign, one azepinone amide hit was found that resembled the native peptide substrate and possessed moderate biochemical potency against three bacterial isozymes. X-ray crystallography was used in combination with substrate-based design to direct the rational optimization of analogs with sub-micromolar potency. The novel compounds presented here represent potent broad-spectrum biochemical inhibitors of bacterial MAP and have the potential to lead to the development of new medicines to combat serious multi-drug resistant infections.

NBTI 5463 is a novel bacterial type II topoisomerase inhibitor with activity against gram-negative bacteria and in vivo efficacy.

The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.

Dimerization of isolated Pseudomonas aeruginosa lipopolysaccharide transporter component LptA.

LptA is a soluble periplasmic component of the lipopolysaccharide (LPS) transport system of Gram-negative bacteria that transports newly synthesized LPS from the inner membrane to the outer leaflet of the outer membrane. LptA links the inner membrane components (LptBFGC) to the outer membrane components (LptDE), but it is uncertain whether LptA is a freely moving LPS shuttle or part of a stable trans-periplasm structure. Escherichiacoli LptA forms highly polymerized head-to-tail oligomers in solution, but dimers in vivo. We studied the oligomerization of purified Pseudomonasaeruginosa LptA. Size-exclusion chromatography showed that P. aeruginosa LptA, unlike E. coli LptA, is a dimer over a wide range of concentrations. Chemical crosslinking with bis(sulfosuccinimidyl) suberate confirmed that dimers were the predominant species even at sub-micromolar LptA concentrations, which was unaffected by LPS binding. Mass spectrometry of crosslinked dimers showed that crosslinks occurred between the N-terminal α-amino group and either Lys-172 or Lys-173 near the C-terminus. These results support a hypothetical structure for the dimer of isolated P. aeruginosa LptA in which the N-terminus of one monomer is in close proximity to the C-terminus of the other, and the same surface of each monomer forms the interface between them, preventing further oligomerization.

A high-throughput fluorescence anisotropy-based assay for human topoisomerase II ß-catalyzed ATP-dependent supercoiled DNA relaxation.

Because of their essentiality for DNA replication, transcription, and repair, type II topoisomerases are targets for antibacterial and anticancer drugs. There are two type II topoisomerases in humans, topoisomerase IIα (TOP2A) and topoisomerase IIß (TOP2B), and two in bacteria, gyrase and topoisomerase IV. Inhibition of one or both of the human type II topoisomerases by antibacterial compounds targeting their bacterial counterparts could result in toxicity. In addition, side effects of anticancer drugs targeting TOP2A could result from inhibition of TOP2B. A simple and rapid biochemical assay for the activity of TOP2A and TOP2B would be advantageous for screening for novel inhibitors, testing them for selectivity for one enzyme over the other, and testing for potential toxicity of antibacterial type II topoisomerases mediated by human topoisomerase II inhibition. In this paper, we show that a previously reported high-throughput, fluorescence anisotropy-based assay for ATP-dependent relaxation of supercoiled DNA by human TOP2A can also be used under identical conditions for human TOP2B. We used this assay to compare the potencies versus both enzymes of 19 compounds reported in the literature to inhibit human and/or bacterial type II topoisomerases. We also used the assay to investigate the effect of ATP concentration on inhibitor potencies.

Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants.

High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial ß-lactam drugs, including penicillins, cephalosporins, and carbapenems. ß-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because ß-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37-43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.

Quantitative investigation of the affinity of human respiratory syncytial virus phosphoprotein C-terminus binding to nucleocapsid protein.

BACKGROUND: There are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N. METHODS: We used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe. RESULTS: Binding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased. CONCLUSIONS: The results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.

Clinical Outcomes for Patients With Monomicrobial vs Polymicrobial Acinetobacter baumannii-calcoaceticus Complex Infections Treated With Sulbactam-Durlobactam or Colistin: A Subset Analysis From a Phase 3 Clinical Trial.

Background: In a previous study, the efficacy and safety of sulbactam-durlobactam vs colistin for the treatment of patients with carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRABC) infections were evaluated in a randomized controlled phase 3 trial. Both arms were dosed on a background of imipenem-cilastatin to treat coinfecting gram-negative pathogens. Thirty-six percent of infections in the primary efficacy population were polymicrobial. Methods: A subset analysis was performed to compare clinical and microbiological outcomes at test of cure (7 ± 2 days after the last dose) for patients with monomicrobial and polymicrobial CRABC infections. Minimal inhibitory concentrations of antibiotics against baseline isolates were determined by broth microdilution according to Clinical and Laboratory Standards Institute methodology. Results: Clinical cure, 28-day all-cause mortality, and microbiological outcomes were similar for patients in the sulbactam-durlobactam treatment arm with monomicrobial or polymicrobial A baumannii-calcoaceticus infections. Patients in the colistin arm with monomicrobial CRABC infections had higher mortality rates with worse clinical and microbiological outcomes as compared with those with polymicrobial infections. For patients who received sulbactam-durlobactam, imipenem susceptibility of coinfecting gram-negative pathogens trended with clinical benefit for patients with polymicrobial A baumannii-calcoaceticus infections. When tested in vitro, durlobactam restored imipenem susceptibility to the majority of coinfecting gram-negative pathogens from the sulbactam-durlobactam arm. This phenotype appeared to be related to the clinical outcome in 13 of 15 evaluable cases. Conclusions: These results suggest that the use of sulbactam-durlobactam plus a carbapenem could be an effective approach to treat polymicrobial infections that include CRABC, but additional clinical data are needed to demonstrate efficacy.

Improvement of the pharmacokinetics and in vivo antibacterial efficacy of a novel type IIa topoisomerase inhibitor by formulation in liposomes.

Several useful properties of liposome-based formulations of various existing antibacterial drugs have been reported. These properties include lower MICs, improved pharmacokinetics, lower toxicity, selective distribution to infected tissues, and enhanced in vivo efficacy. Here we report in vivo studies of a liposomal formulation of a member of a novel class of antibacterial type II topoisomerase inhibitors, others of which have progressed to early phases of clinical trials. The free (i.e., nonliposomal) compound has broad-spectrum MICs but suboptimal pharmacokinetics in rats and mice, characterized by a high volume of distribution and rapid clearance. The liposomal formulation of the compound had essentially unchanged MICs but greatly reduced volume of distribution and clearance in rats and mice. In an in vivo mouse model of Staphylococcus aureus infection of one thigh, the liposomal compound localized preferentially to the infected thigh, whereas the free compound showed no preference for the infected versus the uninfected thigh. Most importantly, the liposomal compound had enhanced efficacy at clearing the infection compared with the free compound. Delivery of this class of compounds as liposomal formulations may offer clinical advantages compared with free compounds.