Penam sulfones and β-lactamase inhibition: SA2-13 and the importance of the C2 side chain length and composition.
β-Lactamases are the major reason β-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, β-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k(react) than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k(react) compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the K→R change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV β-lactamases that possess the K234R substitution.
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, United States of America.
Elizabeth A
EA
Rodkey
Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, United States of America ; Research Division, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, United States of America.
Marisa L
ML
Winkler
Research Division, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, United States of America.
Christopher R
CR
Bethel
Department of Chemistry, Southern Methodist University, Dallas, Texas, United States of America.
Sundar Ram Reddy
SR
Pagadala
Department of Chemistry, Southern Methodist University, Dallas, Texas, United States of America.
John D
JD
Buynak
Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, United States of America ; Research Division, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, United States of America ; Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America ; Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States of America.
Robert A
RA
Bonomo
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, United States of America.
Focco
F
van den Akker
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006574:Heterocyclic Compounds, 2-Ring / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013450:Sulfones / Q000737:chemistry*; D000078142:Tazobactam; D053778:Thiazolidines / Q000737:chemistry*; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006574:Heterocyclic Compounds, 2-Ring; C574280:PSR-3-226; C000591536:SA2-13 compound; D013450:Sulfones; D053778:Thiazolidines; D065093:beta-Lactamase Inhibitors; D010397:Penicillanic Acid; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D000078142:Tazobactam