Serine ?-lactamases are the major resistance mechanism to ?-lactam antibiotics, such as penicillin. In the last 25 years, hundreds of mutant ?-lactamases have appeared in the clinic in response to newer ?-lactams. These enzymes are a pressing medical problem and a fascinating example of molecular evolution. One goal of this research program is to understand the molecular bases of this evolving activity. A second goal is to exploit this information to discover novel inhibitors. Such inhibitors may escape the current cycle of incremental antibiotic modification followed by rapid resistance response.
The specific aims are: 1. To understand the molecular mechanisms of evolved mutant ?-lactamases. Initially introduced to confer stability to ?-lactamases, 3rd generation cephalosporins have promoted the evolution of ?-lactamase mutants. The range of substitutions, and the structural reorganization to which they lead, can be breathtaking. We consider the following questions: a. How do "extended spectrum" substitutions increase activity against once "?-lactamase-stable" cephalosporins? b. How do distant gain-of-function substitutions communicate their effects to the active site? c. Does the activity increase come at a cost to internal stability? We target mutants of the class A and class C ?-lactamases, which are the most widespread. Clinically isolated resistance mutants will be cloned, expressed, their kinetics and stability measured. Their x-ray structures will be determined in complex with substrates and transition-state analogs. These studies will not only inform our understanding of ?-lactamases, but also other resistance enzymes for which they are model systems. 2. To discover novel inhibitors of wild-type and mutant ?-lactamases. Guided by the structural studies of substrate recognition and evolution from aim 1, new ?-lactamase inhibitors will be designed. Both novel molecules, unrelated to ?-lactams, and transition-state analogs will be investigated, using a combination of structure-based design, synthetic elaboration, and crystallography. The following will be explored. a. What is the range of novel chemotypes that can be discovered for ?-lactamase;can these inhibitors be optimized for affinity? b. Optimization of leads from the last period: for transition-state analogs to the sub-nanomolar range, and for novel chemotypes to the sub-micromolar range. c. Will substrate-mimics be more susceptible to pre-evolved resistance mechanisms than the novel inhibitors?

Public Health Relevance

?-lactamases are the major resistance mechanism to penicillin and related antibiotics. In the last 25 years, hundreds of mutant ?-lactamases have appeared in the clinic in response to newer drugs. One goal of this research program is to understand the molecular bases of this evolving activity. A second is to exploit this information to discover novel inhibitors to escape the current cycle of incremental antibiotic modification followed by rapid resistance response.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063815-11
Application #
8232108
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Fabian, Miles
Project Start
2001-08-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
11
Fiscal Year
2012
Total Cost
$281,930
Indirect Cost
$83,930
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Tondi, Donatella; Venturelli, Alberto; Bonnet, Richard et al. (2014) Targeting class A and C serine ?-lactamases with a broad-spectrum boronic acid derivative. J Med Chem 57:5449-58
Minozzi, Manuela; Lattanzi, Gianluca; Benz, Roland et al. (2011) Permeation through the cell membrane of a boron-based ?-lactamase inhibitor. PLoS One 6:e23187
Jadhav, Ajit; Ferreira, Rafaela S; Klumpp, Carleen et al. (2010) Quantitative analyses of aggregation, autofluorescence, and reactivity artifacts in a screen for inhibitors of a thiol protease. J Med Chem 53:37-51
Tondi, Donatella; Calo, Samuele; Shoichet, Brian K et al. (2010) Structural study of phenyl boronic acid derivatives as AmpC beta-lactamase inhibitors. Bioorg Med Chem Lett 20:3416-9
Eidam, Oliv; Romagnoli, Chiara; Caselli, Emilia et al. (2010) Design, synthesis, crystal structures, and antimicrobial activity of sulfonamide boronic acids as ?-lactamase inhibitors. J Med Chem 53:7852-63
Chen, Yu; Zhang, Weilie; Shi, Qicun et al. (2009) Crystal structures of penicillin-binding protein 6 from Escherichia coli. J Am Chem Soc 131:14345-54
Marciano, David C; Pennington, Jeanine M; Wang, Xiaohu et al. (2008) Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 beta-lactamase. J Mol Biol 384:151-64
Morandi, Stefania; Morandi, Federica; Caselli, Emilia et al. (2008) Structure-based optimization of cephalothin-analogue boronic acids as beta-lactamase inhibitors. Bioorg Med Chem 16:1195-205
Chen, Yu; Bonnet, Richard; Shoichet, Brian K (2007) The acylation mechanism of CTX-M beta-lactamase at 0.88 a resolution. J Am Chem Soc 129:5378-80
Venturelli, Alberto; Tondi, Donatella; Cancian, Laura et al. (2007) Optimizing cell permeation of an antibiotic resistance inhibitor for improved efficacy. J Med Chem 50:5644-54

Showing the most recent 10 out of 29 publications