Jed F. Fisher

Recent advances in MMP inhibitor design

The search for an MMP inhibitor with anticancer efficacy is a nearly three-decade endeavor. This inhibitor is yet to be found. The reasons for this failure include shortcomings in the chemistry of these compounds (including broad MMP sub-type selectivity, metabolic lability, and toxicity) as well as the emerging, and arguably extraordinary, complexity of MMP cell (and cancer) biology. Together these suggest that the successful anticancer inhibitor must possess MMP selectivity against the MMP subtype whose involvement is critical, yet highly temporally (with respect to metastatic progression) and mechanistically (with respect to matrix degradation) regulated. This review summarizes the progression of chemical structure and mechanistic thinking toward these objectives, with emphasis on the disappointment, the perseverance, and the resilient optimism that such an inhibitor is there to be discovered.

Bacterial cell-wall recycling

Many Gram‐negative and Gram‐positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. Although cell‐wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell‐wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram‐negative pathogens, anhydro‐MurNAc‐peptide cell‐wall fragments regulate AmpC β‐lactamase induction. In some Gram‐positive organisms, short peptides derived from the cell wall regulate the induction of both β‐lactamase and β‐lactam–resistant penicillin‐binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell‐wall–targeted antibiotics. Indeed, such inhibitors improve the potency of β‐lactams in vitro against inducible AmpC β‐lactamase–producing bacteria. We describe the key steps of cell‐wall remodeling and recycling, the regulation of resistance mechanisms by cell‐wall recycling, and recent advances toward the discovery of cell‐wall–recycling inhibitors.

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.

Development of an immunoassay for endogenous digitalislike factor.

Recently, attempts to purify and identify a circulating inhibitor of the sodium pump have been successful. Based on the outcome of mass spectral analysis of purified inhibitor, we raised in rabbits antibodies to conjugates of the commercially available cardenolide ouabain and used them in the development of an indirect enzyme-linked immunosorbent assay (ELISA) for endogenous digitalislike factor (EDLF). Antisera obtained were of high antibody titer (l:2xlO6) and showed full cross-reactivity with purified EDLF. The antisera were highly specific for ouabain and structurally related cardenolides but showed no cross-reactivity with numerous endogenous steroids and peptides. At each step in the purification of EDLF, inhibition of the sodium pump and immunologic cross-reactivity were inseparable. The ELISA as developed had a working range of 5-2,000 fimol, with an IC50 of 80 fmol/well. Using solid-phase extraction and the ELISA, we determined the circulating level of EDLF in plasma from normal human volunteers to be 138±43 fmol/ml, whereas patients on total parenteral nutrition for at least 1 week had a circulating level of 108 ±17 fmol/ml, suggesting that the circulating factor was of endogenous origin. The ELISA developed appears to measure a naturally occurring counterpart to the cardenolides that could play a role in modulating the sodium pump and thereby cellular electrolyte homeostasis. (Hypertension 1991;17:936-943)

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.

Inhibition of Histone Deacetylases: A Pharmacological Approach to the Treatment of Non-Cancer Disorders

The dynamics of gene expression are regulated by histone acetylases (HATs) and histone deacetylases (HDACs) that control the acetylation state of lysine side chains of the histone proteins of chromatin. The catalytic activity of these two enzymes remodels chromatin to control gene expression without altering gene sequence. Treatment of cancer has been the primary target for the clinical development of HDAC inhibitors, culminating in approval for the first HDAC inhibitor for the treatment of cutaneous T cell lymphoma. Beyond cancer, HDAC inhibition has potential for the treatment of many other diseases. The HDAC inhibitors phenylbutyric acid, valproic acid, and suberoylanilide hydroxamic acid (SAHA) have been shown to correct errant gene expression, ameliorate the progression of disease, and restore absent synthetic or metabolic activities for a diverse group of non-cancer disorders. These benefits have been found in patients with sickle cell anemia, HIV, and cystic fibrosis. In vitro and in vivo models of spinal muscular atrophy, muscular dystrophy, and neurodegenerative, and inflammatory disorders also show response to HDAC inhibitors. This review examines the application of HDAC inhibition as a treatment for a wide-range of non-cancer disorders, many of which are rare diseases that urgently need therapy. Inhibition of the HDACs has general potential as a pharmacological epigenetic approach for gene therapy.

Messenger Functions of the Bacterial Cell Wall-derived Muropeptides

Bacterial muropeptides are soluble peptidoglycan structures central to recycling of the bacterial cell wall and messengers in diverse cell signaling events. Bacteria sense muropeptides as signals that antibiotics targeting cell-wall biosynthesis are present, and eukaryotes detect muropeptides during the innate immune response to bacterial infection. This review summarizes the roles of bacterial muropeptides as messengers, with a special emphasis on bacterial muropeptide structures and the relationship of structure to the biochemical events that the muropeptides elicit. Muropeptide sensing and recycling in both Gram-positive and Gram-negative bacteria are discussed, followed by muropeptide sensing by eukaryotes as a crucial event in the innate immune response of insects (via peptidoglycan-recognition proteins) and mammals (through Nod-like receptors) to bacterial invasion.

Matrix Metalloproteinase 2 Inhibition: Combined Quantum Mechanics and Molecular Mechanics Studies of the Inhibition Mechanism of (4-Phenoxyphenylsulfonyl)methylthiirane and Its Oxirane Analogue

The inhibition mechanism of matrix metalloproteinase 2 (MMP2) by the selective inhibitor (4-phenoxyphenylsulfonyl)methylthiirane (SB-3CT) and its oxirane analogue is investigated computationally. The inhibition mechanism involves C-H deprotonation with concomitant opening of the three-membered heterocycle. SB-3CT was docked into the active site of MMP2, followed by molecular dynamics simulation to prepare the complex for combined quantum mechanics and molecular mechanics (QM/MM) calculations. QM/MM calculations with B3LYP/6-311+G(d,p) for the QM part and the AMBER force field for the MM part were used to examine the reaction of these two inhibitors in the active site of MMP2. The calculations show that the reaction barrier for transformation of SB-3CT is 1.6 kcal/mol lower than its oxirane analogue, and the ring-opening reaction energy of SB-3CT is 8.0 kcal/mol more exothermic than that of its oxirane analogue. Calculations also show that protonation of the ring-opened product by water is thermodynamically much more favorable for the alkoxide obtained from the oxirane than for the thiolate obtained from the thiirane. A six-step partial charge fitting procedure is introduced for the QM/MM calculations to update atomic partial charges of the quantum mechanics region and to ensure consistent electrostatic energies for reactants, transition states, and products.

Three Decades of the Class A β-Lactamase Acyl-Enzyme

The discovery that the mechanism of beta-lactam hydrolysis catalyzed by the class A (active site serine-dependent) beta-lactamases proceeds via an acyl-enzyme intermediate was made thirty years ago. Since this discovery, the active site circumstance that enables acylation of the active site serine and further enables hydrolytic deacylation of the acyl-serine intermediate, has received extraordinary scrutiny. The justification for this scrutiny is the direct relevance of the beta-lactamases to the manifestation of bacterial resistance to the beta-lactam antibiotics, and the subsequent (to the discovery of the beta-lactamase acyl-enzyme) recognition of the direct evolutionary relationship between the serine beta-lactamase acyl-enzyme, and the penicillin binding protein acyl-enzyme that is key to beta-lactam antibiotic activity. This short review describes the early events leading to the recognition that serine beta-lactamase catalysis proceeds via an acyl-enzyme intermediate, and summarizes several of the key mechanistic studies--including infrared spectroscopy, cryoenzymology, beta-lactam design, and x-ray crystallography--that have been exploited to understand this pivotal catalytic intermediate.