DISCOVERY OF NEW ANTIMICROBIAL OPTIONS AND EVALUATION OF AMINOGLYCOSIDE RESISTANCE ENZYME-ASSOCIATED RESISTANCE EPIDEMIC

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6\u27) domain and its mutant enzymes from a bifunctional AME, AAC(6\u27)-Ie/APH(2 )-Ia as well as another common AME, APH(3\u27)-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi

Expanding Aminoglycoside Resistance Enzyme Regiospecificity by Mutation and Truncation

Aminoglycosides (AGs) are broad-spectrum antibiotics famous for their antibacterial activity against Gram-positive and Gram-negative bacteria, as well as mycobacteria. In the United States, the most prescribed AGs, including amikacin (AMK), gentamicin (GEN), and tobramycin (TOB), are vital components of the treatment for resistant bacterial infections. Arbekacin (ABK), a semisynthetic AG, is widely used for the treatment of resistant <i>Pseudomonas aeruginosa</i> and methicillin-resistant <i>Staphylococcus aureus</i> in Asia. However, the rapid emergence and development of bacterial resistance are limiting the clinical application of AG antibiotics. Of all bacterial resistance mechanisms against AGs, the acquisition of AG-modifying enzymes (AMEs) by bacteria is the most common. It was previously reported that a variant of a bifunctional AME, the 6′-<i>N</i>-AG acetyltransferase-Ie/2″-<i>O</i>-AG phosphotransferase-Ia [AAC(6′)-Ie/APH(2″)-Ia], containing a D80G point mutation and a truncation after amino acid 240 modified ABK and AMK at a new position, the 4‴-amine, therefore displaying a change in regiospecificity. In this study, we aimed to verify the altered regiospecificity of this bifunctional enzyme by mutation and truncation for the potential of derivatizing AGs with chemoenzymatic reactions. With the three variant enzymes in this study that contained either mutation only (D80G), truncation only (1–240), or mutation and truncation (D80G-1–240), we characterized their activity by profiling their substrate promiscuity, determined their kinetics parameters, and performed mass spectrometry to determine how and where ABK and AMK were acetylated by these enzymes. We found that the three mutant enzymes possessed distinct acetylation regiospecificity compared to that of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme and the functional AAC(6′)-Ie domain [AAC(6′)/APH(2″)-1–194]

Combating Enhanced Intracellular Survival (Eis)-Mediated Kanamycin Resistance of Mycobacterium tuberculosis by Novel Pyrrolo[1,5‑<i>a</i>]pyrazine-Based Eis Inhibitors

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. Hence, the identification of highly effective antitubercular drugs with novel modes of action is crucial. In this paper, we report the discovery and development of pyrrolo­[1,5-<i>a</i>]­pyrazine-based analogues as highly potent inhibitors of the Mycobacterium tuberculosis (<i>Mtb</i>) acetyltransferase enhanced intracellular survival (Eis), whose up-regulation causes clinically observed resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN). We performed a structure–activity relationship (SAR) study to optimize these compounds as potent Eis inhibitors both against purified enzyme and in mycobacterial cells. A crystal structure of Eis in complex with one of the most potent inhibitors reveals that the compound is bound to Eis in the AG binding pocket, serving as the structural basis for the SAR. These Eis inhibitors have no observed cytotoxicity to mammalian cells and are promising leads for the development of innovative AG adjuvant therapies against drug-resistant TB