Leticia I. Llarrull

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function.

Significance Penicillin binding protein 2a imparts to the human pathogen Staphylococcus aureus resistance to β-lactam antibiotics. Our structural characterization of the allosteric basis governing its resistance mechanism identifies a basis for the design of new antibacterials that can both activate and inhibit this key resistance enzyme. The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd-transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.

The future of the β-lactams.

In the 80 years since their discovery the β-lactam antibiotics have progressed through structural generations, each in response to the progressive evolution of bacterial resistance mechanisms. The generational progression was driven by the ingenious, but largely empirical, manipulation of structure by medicinal chemists. Nonetheless, the true creative force in these efforts was Nature, and as the discovery of new β-lactams from Nature has atrophied while at the same time multi-resistant and opportunistic bacterial pathogens have burgeoned, the time for empirical drug discovery has passed. We concisely summarize recent developments with respect to bacterial resistance, the identity of the new β-lactams, and the emerging non-empirical strategies that will ensure that this incredible class of antibiotics has a future.

Molecular Basis and Phenotype of Methicillin Resistance in Staphylococcus aureus and Insights into New β-Lactams That Meet the Challenge

The gram-positive bacterium Staphylococcus aureus is a leading cause of hospital- and community-associated infections (16, 85, 108). In the hospital, S. aureus is the most frequent cause of surgical, lower respiratory tract, and cardiovascular infections. Furthermore, it is the second most common cause of health care-associated pneumonia and bloodstream infections (108, 151, 152). Historically, β-lactam antibiotics have exhibited potent activity against S. aureus, which along with good safety profiles make them the agents of choice for the treatment of staphyloccocal infections. Of particular concern now is the growing prevalence of methicillin (meticillin)-resistant S. aureus (MRSA) in both hospital- and community-associated infections (24, 70, 133). The development of resistance to β-lactam antimicrobials, often concurrently with resistance to other antimicrobial agents, poses a great challenge to the prevention and treatment of S. aureus infections (7, 108). Staphylococci have two primary mechanisms for resistance to β-lactam antibiotics: the expression of an enzyme (the PC1 β-lactamase) capable of hydrolyzing the β-lactam ring, thus rendering the antibiotic inactive, and the acquisition of a gene encoding a modified penicillin-binding protein (PBP), known as PBP 2a, found in MRSA and coagulase-negative staphylococci. PBP 2a is intrinsically resistant to inhibition by β-lactams (59). PBP 2a remains active in the presence of concentrations of β-lactam antibiotics that inhibit most endogenous PBP enzymes, thus substituting for their functions in cell wall synthesis and allowing growth in the presence of the β-lactam inhibitors. This review briefly discusses the structure and synthesis of the S. aureus cell wall, the resistance to β-lactam antibiotics through the acquisition of PBP 2a, the evolution of MRSA, and the involvement of other protein factors in methicillin resistance. In addition, the characteristics of new β-lactam antibiotics that target PBP 2a are discussed, along with their role as important new entities in the antibacterial pipeline for the treatment of MRSA infections.

Discovery of a New Class of Non-β-lactam Inhibitors of Penicillin-Binding Proteins with Gram-Positive Antibacterial Activity

Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-β-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin- and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.

Trapping and Characterization of a Reaction Intermediate in Carbapenem Hydrolysis by B. cereus Metallo-β-lactamase

Metallo-beta-lactamases hydrolyze most beta-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem in both the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the beta-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species with a novel resonant structure that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2, and B3 lactamases, identification of this intermediate could be exploited as a first step toward the design of transition-state-based inhibitors for all three classes of metallo-beta-lactamases.

Metal Content and Localization during Turnover in B. cereus Metallo-β-lactamase

Metallo-beta-lactamases are enzymes capable of hydrolyzing all known classes of beta-lactam antibiotics, rendering them ineffective. The design of inhibitors active against all classes of metallo-beta-lactamases has been hampered by the heterogeneity in metal content in the active site and the existence of two different mononuclear forms. BcII is a B1 metallo-beta-lactamase which is found in both mononuclear and dinuclear forms. Despite very elegant studies, there is still controversy on the nature of the active BcII species. We carried out a non-steady-state study of the hydrolysis of penicillin G catalyzed by Co(II)-substituted BcII, and we followed the modifications occurring at the active site of the enzyme. Working at different metal/enzyme ratios we demonstrate that both mono-Co(II) and di-Co(II) BcII are active metallo-beta-lactamases. Besides, we here present evidence that during penicillin G hydrolysis catalyzed by mono-Co(II) BcII the metal is localized in the DCH site (the Zn2 site in B1 enzymes). These conclusions allow us to propose that both in mono-Co(II) and di-Co(II) BcII the substrate is bound to the enzyme through interactions with the Co(II) ion localized in the DCH site. The finding that the DCH site is able to give rise to an active lactamase suggests that the Zn2 site is a common feature to all subclasses of metallo-beta-lactamases and would play a similar role. This proposal provides a starting point for the design of inhibitors based on transition-state analogs, which might be effective against all MbetaLs.

Reactions of all Escherichia coli lytic transglycosylases with bacterial cell wall.

The reactions of all seven Escherichia coli lytic transglycosylases with purified bacterial sacculus are characterized in a quantitative manner. These reactions, which initiate recycling of the bacterial cell wall, exhibit significant redundancy in the activities of these enzymes along with some complementarity. These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall.

Activation of BlaR1 Protein of Methicillin-resistant Staphylococcus aureus, Its Proteolytic Processing, and Recovery from Induction of Resistance

Background: Resistance to β-lactam antibiotics is regulated by the bla operon. Results: The fate of BlaR1, BlaI, and β-lactamase in the course of induction of resistance is evaluated. Conclusion: BlaR1 fragments in a specific way to allow recovery from induction of resistance. Significance: The processes of induction of resistance and recovery from it have been explained. The fates of BlaI, the gene repressor protein for the bla operon, BlaR1, the β-lactam sensor/signal transducer, and PC1 β-lactamase in four strains of Staphylococcus aureus upon exposure to four different β-lactam antibiotics were investigated as a function of time. The genes for the three proteins are encoded by the bla operon, the functions of which afford inducible resistance to β-lactam antibiotics in S. aureus. BlaR1 protein is expressed at low copy number. Acylation of the sensor domain of BlaR1 by β-lactam antibiotics initiates signal transduction to the cytoplasmic domain, a zinc protease, which is activated and degrades BlaI. This proteolytic degradation derepresses transcription of all three genes, resulting in inducible resistance. These processes take place within minutes of exposure to the antibiotics. The BlaR1 protein was shown to undergo fragmentation in three S. aureus strains within the time frame relevant for manifestation of resistance and was below the detection threshold in the fourth. Two specific sites of fragmentation were identified, one cytoplasmic and the other in the sensor domain. This is proposed as a means for turnover, a process required for recovery from induction of resistance in S. aureus in the absence of the antibiotic challenge. In S. aureus not exposed to β-lactam antibiotics (i.e. not acylated by antibiotic) the same fragmentation of BlaR1 is still observed, including the shedding of the sensor domain, an observation that leads to the conclusion that the sites of proteolysis might have evolved to predispose the protein to degradation within a set period of time.

Active Site Ring-Opening of a Thiirane Moiety and Picomolar Inhibition of Gelatinases

(±)‐2‐[(4‐Phenoxyphenylsulfonyl)methyl]thiirane 1 is a potent and selective mechanism‐based inhibitor of the gelatinase sub‐class of the zinc‐dependent matrix metalloproteinase family. Inhibitor 1 has excellent activity in in vivo models of gelatinase‐dependent disease. We demonstrate that the mechanism of inhibition is a rate‐limiting gelatinase‐catalyzed thiolate generation via deprotonation adjacent to the thiirane, with concomitant thiirane opening. A corollary to this mechanism is the prediction that thiol‐containing structures, related to thiirane‐opened 1, will possess potent matrix metalloproteinase inhibitory activity. This prediction was validated by the synthesis of the product of this enzyme‐catalyzed reaction on 1, which exhibited a remarkable Ki of 530 pm against matrix metalloproteinase‐2. Thiirane 1 acts as a caged thiol, unmasked selectively in the active sites of gelatinases. This mechanism is unprecedented in the substantial literature on inhibition of zinc‐dependent hydrolases.

High-Resolution Crystal Structure of Mlte, an Outer Membrane-Anchored Endolytic Peptidoglycan Lytic Transglycosylase from Escherichia Coli.

The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane.