Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Christopher Davies

Second Advisor

Patrick Woster

Third Advisor

Shaun Olsen

Fourth Advisor

John J. Lemasters

Fifth Advisor

Roger White

Abstract

Gonorrhea is the second most common sexually transmitted bacterial infection in the United States, with nearly 600,000 cases reported in 2018 by the Centers for Disease Control. Alarmingly, the causative agent Neisseria gonorrhoeae has developed resistance to a number of antimicrobials over the last century. With limited options remaining, the CDC now recommends dual therapy with ceftriaxone and azithromycin to decrease the likelihood of resistance development. However, strains with combined cephalosporin and macrolide resistance have now emerged, raising concerns of a post-antibiotic future in which untreatable gonorrhea would impose enormous human and economic cost. The discovery and development of novel antigonococcal agents is, therefore, necessary to avoid a public health crisis. The pharmacologic receptors for β-lactams are a group of transpeptidases known as penicillin-binding proteins (PBP), which catalyze the cross-linkage of peptidoglycan, an essential component of the bacterial cell wall that plays major roles in cell growth and division. N. gonorrhoeae develops chromosomally mediated β-lactam resistance via alterations of PBPs affecting drug affinity, specifically through the acquisition of mutations in the penA gene encoding PBP2. Resistant strains harbor mosaic penA alleles encoding PBP2 variants containing around 60 amino acid changes compared to wild-type. In this work, we examine inhibition of a mosaic form of N. gonorrhoeae PBP2 from the cephalosporin-resistant strain H041, seeking to understand better which features of ligand structure enhance or diminish PBP2 binding in order to develop more effective PBP2 inhibitors. First, we report structure-activity relationships (SAR) for the cephalosporin class of β-lactams against PBP2 from N. gonorrhoeae H041 with the goal of identifying or designing cephalosporins effective against resistant strains. We find that structural features of the C7 acylamino side chain (R1) correlate highly with the second-order rate of PBP2H041 acylation, including increased size, modest lipophilicity, and two ring systems separated by a single branch point. The C3 side chain (R2) makes lesser, but still important, contributions to inhibition, with electronegative elements and planarity enhancing activity. We also found that many of the features enhancing target inhibition (e.g., lipophilicity, aromaticity) diminish antimicrobial activity against the H041 strain, perhaps due to decreased accumulation in the periplasm. Finally, we identify cefoperazone as highly active against PBP2H041 and similarly active against N. gonorrhoeae H041 both in vitro and in vivo compared to ceftriaxone. Second, we report the in silico discovery of novel noncovalent PBP2 inhibitors possessing a 1,1’-biphenyl system. Arylamide JEK-42 and its isosteric sulfonamide derivative JMT-1 are capable of inhibiting PBP2 from both β-lactam-susceptible and -resistant gonococcal strains. Their cross-inhibition of P. aeruginosa PBP3, predicted binding modes showing interaction with highly conserved residues, and structural similarities to bicyclic β-lactam scaffolds indicate their potential for broader activity against class B PBPs. Using the structural similarities between JEK-42, JMT-1, and bicyclic β-lactam scaffolds (i.e., penam, carbapenem, and cephem), a three-point pharmacophore was generated that can be used to identify additional PBP-inhibitory scaffolds. Third, we report the synthesis of 127 derivatives of JMT-1, showing specific substitutions that enhance the inhibition of PBP2 derived from both β-lactam-susceptible and -resistant strains. In keeping with the cephalosporin SAR, hydrophobic substitutions enhance PBP2 inhibition, likely through increased van der Waals contact with the active site, but they can also result in diminished antimicrobial activity. Together, our efforts yielded 10 compounds that show near full inhibition of PBP2 from susceptible and resistant strains, as well as large zones of gonococcal growth inhibition in disc diffusion assays. These studies lay the groundwork for the development of several structurally diverse antigonococcal chemotypes, thereby increasing the probability of producing a successful preclinical candidate.

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