Aminomethyl Spectinomycins as Therapeutics for Drug-Resistant Gonorrhea and Chlamydia Coinfections.

Bacterial sexually transmitted infections are widespread and common, with Neisseria gonorrhoeae (gonorrhea) and Chlamydia trachomatis (chlamydia) being the two most frequent causes. If left untreated, both infections can cause pelvic inflammatory disease, infertility, ectopic pregnancy, and other sequelae. The recommended treatment for gonorrhea is ceftriaxone plus azithromycin (to empirically treat chlamydial coinfections). Antibiotic resistance to all existing therapies has developed in gonorrheal infections. The need for new antibiotics is great, but the pipeline for new drugs is alarmingly small. The aminomethyl spectinomycins, a new class of semisynthetic analogs of the antibiotic spectinomycin, were developed on the basis of a computational analysis of the spectinomycin binding site of the bacterial 30S ribosome and structure-guided synthesis. The compounds display particular potency against common respiratory tract pathogens as well as the sexually transmitted pathogens that cause gonorrhea and chlamydia. Here, we demonstrate the in vitro potencies of several compounds of this class against both bacterial species; the compounds displayed increased potencies against N. gonorrhoeae compared to that of spectinomycin and, significantly, demonstrated activity against C. trachomatis that is not observed with spectinomycin. Efficacies of the compounds were compared to those of spectinomycin and gentamicin in a murine model of infection caused by ceftriaxone/azithromycin-resistant N. gonorrhoeae; the aminomethyl spectinomycins significantly reduced the colonization load and were as potent as the comparator compounds. In summary, data produced by this study support aminomethyl spectinomycins as a promising replacement for spectinomycin and antibiotics such as ceftriaxone for treating drug-resistant gonorrhea, with the added benefit of treating chlamydial coinfections.

Detecting a quasi-stable imine species on the reaction pathway of SHV-1 ß-lactamase and 6ß-(hydroxymethyl)penicillanic acid sulfone.

For the class A ß-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6ß position by a CH2OH group (6ß-(hydroxymethyl)penicillanic acid). The 6ß substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a ß-aminoacrylate) in 1 h for sulbactam and in 4 h for 6ß-(hydroxymethyl) sulbactam. Rapid mix-rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6ß-(hydroxymethyl) sulbactam formed longer-lived acyl-enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6ß-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6ß-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis.

Triethysilyl Enol Ethers in the Synthesis of Carbapenem Precursors.

A diastereoselective process for the formation of intermediates suitable for the preparation of C1 substituted carbapenems was developed. The process is readily scalable and does not involve organometallics or strong bases such as LDA.

ß-Lactamase inhibition by 7-alkylidenecephalosporin sulfones: allylic transposition and formation of an unprecedented stabilized acyl-enzyme.

The inhibition of the class A SHV-1 ß-lactamase by 7-(tert-butoxycarbonyl)methylidenecephalosporin sulfone was examined kinetically, spectroscopically, and crystallographically. An 1.14 Å X-ray crystal structure shows that the stable acyl-enzyme, which incorporates an eight-membered ring, is a covalent derivative of Ser70 linked to the 7-carboxy group of 2-H-5,8-dihydro-1,1-dioxo-1,5-thiazocine-4,7-dicarboxylic acid. A cephalosporin-derived enzyme complex of this type is unprecedented, and the rearrangement leading to its formation may offer new possibilities for inhibitor design. The observed acyl-enzyme derives its stability from the resonance stabilization conveyed by the ß-aminoacrylate (i.e., vinylogous urethane) functionality as there is relatively little interaction of the eight-membered ring with active site residues. Two mechanistic schemes are proposed, differing in whether, subsequent to acylation of the active site serine and opening of the ß-lactam, the resultant dihydrothiazine fragments on its own or is assisted by an adjacent nucleophilic atom, in the form of the carbonyl oxygen of the C7 tert-butyloxycarbonyl group. This compound was also found to be a submicromolar inhibitor of the class C ADC-7 and PDC-3 ß-lactamases.

Early insights into the interactions of different ß-lactam antibiotics and ß-lactamase inhibitors against soluble forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3.

Acinetobacter baumannii is an increasingly problematic pathogen in United States hospitals. Antibiotics that can treat A. baumannii are becoming more limited. Little is known about the contributions of penicillin binding proteins (PBPs), the target of ß-lactam antibiotics, to ß-lactam-sulbactam susceptibility and ß-lactam resistance in A. baumannii. Decreased expression of PBPs as well as loss of binding of ß-lactams to PBPs was previously shown to promote ß-lactam resistance in A. baumannii. Using an in vitro assay with a reporter ß-lactam, Bocillin, we determined that the 50% inhibitory concentrations (IC(50)s) for PBP1a from A. baumannii and PBP3 from Acinetobacter sp. ranged from 1 to 5 µM for a series of ß-lactams. In contrast, PBP3 demonstrated a narrower range of IC(50)s against ß-lactamase inhibitors than PBP1a (ranges, 4 to 5 versus 8 to 144 µM, respectively). A molecular model with ampicillin and sulbactam positioned in the active site of PBP3 reveals that both compounds interact similarly with residues Thr526, Thr528, and Ser390. Accepting that many interactions with cell wall targets are possible with the ampicillin-sulbactam combination, the low IC(50)s of ampicillin and sulbactam for PBP3 may contribute to understanding why this combination is effective against A. baumannii. Unraveling the contribution of PBPs to ß-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel ß-lactams.

C6 Hydroxymethyl-Substituted Carbapenem MA-1-206 Inhibits the Major Acinetobacter baumannii Carbapenemase OXA-23 by Impeding Deacylation.

Acinetobacter baumannii has become a major nosocomial pathogen, as it is often multidrug-resistant, which results in infections characterized by high mortality rates. The bacterium achieves high levels of resistance to ß-lactam antibiotics by producing ß-lactamases, enzymes which destroy these valuable agents. Historically, the carbapenem family of ß-lactam antibiotics have been the drugs of choice for treating A. baumannii infections. However, their effectiveness has been significantly diminished due to the pathogen's production of carbapenem-hydrolyzing class D ß-lactamases (CHDLs); thus, new antibiotics and inhibitors of these enzymes are urgently needed. Here, we describe a new carbapenem antibiotic, MA-1-206, in which the canonical C6 hydroxyethyl group has been replaced with hydroxymethyl. The antimicrobial susceptibility studies presented here demonstrated that this compound is more potent than meropenem and imipenem against A. baumannii producing OXA-23, the most prevalent CHDL of this pathogen, and also against strains producing the CHDL OXA-24/40 and the class B metallo-ß-lactamase VIM-2. Our kinetic and mass spectrometry studies revealed that this drug is a reversible inhibitor of OXA-23, where inhibition takes place through a branched pathway. X-ray crystallographic studies, molecular docking, and molecular dynamics simulations of the OXA-23-MA-1-206 complex show that the C6 hydroxymethyl group forms a hydrogen bond with the carboxylated catalytic lysine of OXA-23, effectively preventing deacylation. These results provide a promising strategy for designing a new generation of CHDL-resistant carbapenems to restore their efficacy against deadly A. baumannii infections. IMPORTANCE Carbapenem antibiotics are the drugs of choice for treatment of deadly infections caused by Gram-negative bacteria. However, their efficacy is severely compromised by the wide spread of carbapenem-hydrolyzing class D ß-lactamases (CHDLs). The importance of this research is the discovery that substitution of the canonical hydroxyethyl group of carbapenems by a hydroxymethyl significantly enhances stability against inactivation by the major CHDL of Acinetobacter baumannii, OXA-23. These results provide a novel strategy for designing next-generation, carbapenemase-stable carbapenems to fight multidrug-resistant infections caused by Gram-negative pathogens.

Atypically Modified Carbapenem Antibiotics Display Improved Antimycobacterial Activity in the Absence of ß-Lactamase Inhibitors.

Commercial carbapenem antibiotics are being used to treat multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis. Like other ß-lactams, carbapenems are irreversible inhibitors of serine d,d-transpeptidases involved in peptidoglycan biosynthesis. In addition to d,d-transpeptidases, mycobacteria also utilize nonhomologous cysteine l,d-transpeptidases (Ldts) to cross-link the stem peptides of peptidoglycan, and carbapenems form long-lived acyl-enzymes with Ldts. Commercial carbapenems are C2 modifications of a common scaffold. This study describes the synthesis of a series of atypical, C5α modifications of the carbapenem scaffold, microbiological evaluation against Mycobacterium tuberculosis (Mtb) and the nontuberculous mycobacterial species, Mycobacterium abscessus (Mab), as well as acylation of an important mycobacterial target Ldt, LdtMt2. In vitro evaluation of these C5α-modified carbapenems revealed compounds with standalone (i.e., in the absence of a ß-lactamase inhibitor) minimum inhibitory concentrations (MICs) superior to meropenem-clavulanate for Mtb, and meropenem-avibactam for Mab. Time-kill kinetics assays showed better killing (2-4 log decrease) of Mtb and Mab with lower concentrations of compound 10a as compared to meropenem. Although susceptibility of clinical isolates to meropenem varied by nearly 100-fold, 10a maintained excellent activity against all Mtb and Mab strains. High resolution mass spectrometry revealed that 10a acylates LdtMt2 at a rate comparable to meropenem, but subsequently undergoes an unprecedented carbapenem fragmentation, leading to an acyl-enzyme with mass of Δm = +86 Da. Rationale for the divergence of the nonhydrolytic fragmentation of the LdtMt2 acyl-enzymes is proposed. The observed activity illustrates the potential of novel atypical carbapenems as prospective candidates for treatment of Mtb and Mab infections.

Aminomethyl spectinomycins: a novel antibacterial chemotype for biothreat pathogens.

New antibiotics that are active against multi-drug-resistant strains and difficult-to-treat bacterial infections are needed. Synthetic modification of spectinomycin, a bacterial protein synthesis inhibitor, has been shown to increase antibacterial activity compared with spectinomycin. Aminomethyl spectinomycins are active against Gram-negative and Gram-positive bacterial pathogens. In this study, the ability of aminomethyl spectinomycins to treat biothreat pathogens is examined by MIC profiling, synergy testing, and in vivo efficacy experiments. Compound 1950 exhibited potent antibacterial activity against Gram-negative pathogens Brucella spp., Burkholderia mallei, and Francisella tularensis, but showed little to no growth inhibition against Burkholderia pseudomallei, Bacillus anthracis, and Yersinia pestis. Combination testing in checkerboard assays revealed that aminomethyl spectinomycin-antibiotic combinations had mainly an additive effect against the susceptible biodefense pathogens. The in vivo efficacy of compound 1950 was also demonstrated in mice infected with B. mallei (FMH) or F. tularensis (SchuS4). These results suggest that aminomethyl spectinomycins are promising new candidates for development of therapeutics against biodefense bacterial agents.