The emergence and dissemination of carbapenem-resistant Gram-negative pathogens is a significant source of healthcare-associated morbidity and mortality. Carbapenemases (i.e., carbapenem-hydrolyzing ?-lactamases) that potentially inactivate all ?-lactams severely limit the antimicrobials available to treat infections with these bacteria. Presently, two major carbapenemases are emerging in Klebsiella and are a major threat to our antibiotic armamentarium: the Klebsiella pneumoniae carbapenemase (KPC-2), and the New Delhi Metallo-?-lactamase (NDM-1 MBL). KPC is a class A carbapenemase that hydrolyzes imipenem, ertapenem, meropenem, doripenem, and ?- lactamase inhibitors and is found in almost every major tertiary care facility in the US. In order to design future ?-lactams to combat these imminent threats, we need to understand how the substrate profile and susceptibility patterns of strains that possess KPC and NDM-1 will change as a result of substitutions in """"""""hot spots"""""""" in the ?-lactamase (i.e., &-loop, amino acid residues 164 to 179 in KPC-2, and the loop regions and entrance to the active site of NDM-1). These challenges frame the research direction that will be addressed herein. The major research questions we will ask in this Merit Proposal are: 1) If substitutions in the &-loop arise in KPC-2, will they pose a significant clincal threat by representing novel phenotypes that will present even greater challenges to the clinician? 2) How do unique structural features of NDM-1 MBL impact the ability of the ?-lactamase to hydrolyze ?-lactams? To answer the first question our plan is to determine the effect of substitutions in the &-loop at amino acid positions R164, E166, N170, and D179 in KPC-2 and ascertain how these mutations alter microbiological phenotype, protein stability and catalytic mechanism. To address the second question we will use mutagenesis to remove, in a stepwise manner, the """"""""distinctive additional sequence"""""""" at amino acid positions 163 to 166 ( 163-166) in NDM-1 and assess the impact of these deletions on resistance (phenotype), hydrolysis of standard ?-lactam and carbapenem substrates, and protein stability. Next, we will use mutagenesis to replace (site-saturation) and to delete F70 ( 70) in NDM-1, an amino acid at the entrance of the active site and assess the activity of this variant and its role in protein stability and catalysis. By focusing on the carbapenemases of K. pneumoniae we will provide the critical knowledge that leads to novel therapies. Our laboratory is actively defining the amino acid sequence requirements of KPC-2 and this new project will stand out as an important undertaking in the further exploration of the role of the &-loop and in the biochemical study of NDM-1. Achieving our objectives will advance the general understanding needed for the medicinal chemist to design effective therapeutic agents that will act as substrates and inhibitors against these """"""""versatile"""""""" ?-lactamases and help us devise antibiotic treatment regimens that will assist physicians faced with this serious and emerging infectious disease threats.
Antibiotic-resistant infections are rapidly spreading and taking a staggering toll on all healthcar systems; including the VA. Nearly 2 million Americans per year develop hospital-acquired infections; resulting in 99;000+ deaths - the vast majority of which are due to antibacterial-resistant Gram- negative pathogens. Of critical importance; is the fact that there are few to no approved antibacterial drugs currently available to treat many Gram-negative bacterial infections. The two greatest threats to our antibiotic armamentarium are the carbapenem hydrolyzing -lactamases; KPC and NDM-1; found in Klebsiella pneumoniae. In an effort to find new therapies; our laboratory will attempt to identify the essential structure/function relationships present in these two carbapenemases that are important in catalysis to provide the design principles for novel inhibitors.
|El Chakhtoura, Nadim G; Saade, Elie; Iovleva, Alina et al. (2018) Therapies for multidrug resistant and extensively drug-resistant non-fermenting gram-negative bacteria causing nosocomial infections: a perilous journey toward 'molecularly targeted' therapy. Expert Rev Anti Infect Ther 16:89-110|
|Nukaga, Michiyoshi; Papp-Wallace, Krisztina M; Hoshino, Tyuji et al. (2018) Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam. Antimicrob Agents Chemother 62:|
|Kanwar, Anubhav; Marshall, Steven H; Perez, Federico et al. (2018) Emergence of Resistance to Colistin During the Treatment of Bloodstream Infection Caused by Klebsiella pneumoniae Carbapenemase-Producing Klebsiella pneumoniae. Open Forum Infect Dis 5:ofy054|
|Ghiglione, Barbara; Rodríguez, María Margarita; Curto, Lucrecia et al. (2018) Defining Substrate Specificity in the CTX-M Family: the Role of Asp240 in Ceftazidime Hydrolysis. Antimicrob Agents Chemother 62:|
|Papp-Wallace, Krisztina M; Barnes, Melissa D; Alsop, Jim et al. (2018) Relebactam Is a Potent Inhibitor of the KPC-2 ?-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae. Antimicrob Agents Chemother 62:|
|Tsalik, Ephraim L; Bonomo, Robert A; Fowler Jr, Vance G (2018) New Molecular Diagnostic Approaches to Bacterial Infections and Antibacterial Resistance. Annu Rev Med 69:379-394|
|Cheng, Zishuo; VanPelt, Jamie; Bergstrom, Alexander et al. (2018) A Noncanonical Metal Center Drives the Activity of the Sediminispirochaeta smaragdinae Metallo-?-lactamase SPS-1. Biochemistry 57:5218-5229|
|Papp-Wallace, Krisztina M; Nguyen, Nhu Q; Jacobs, Michael R et al. (2018) Strategic Approaches to Overcome Resistance against Gram-Negative Pathogens Using ?-Lactamase Inhibitors and ?-Lactam Enhancers: Activity of Three Novel Diazabicyclooctanes WCK 5153, Zidebactam (WCK 5107), and WCK 4234. J Med Chem 61:4067-4086|
|Bahr, Guillermo; Vitor-Horen, Luisina; Bethel, Christopher R et al. (2018) Clinical Evolution of New Delhi Metallo-?-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation. Antimicrob Agents Chemother 62:|
|Bergstrom, Alexander; Katko, Andrew; Adkins, Zach et al. (2018) Probing the Interaction of Aspergillomarasmine A with Metallo-?-lactamases NDM-1, VIM-2, and IMP-7. ACS Infect Dis 4:135-145|
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