C4-Phenylthio ß-lactams: Effect of the chirality of the ß-lactam ring on antimicrobial activity.

C4-phenylthio ß-lactams are a new family of antibacterial agents that have activity against two phylogenetically distant bacteria - Mycobacterium tuberculosis (Mtb) and Moraxella catarrhalis (M. cat). These compounds are effective against ß-lactamase producing Mtb and M. cat unlike the clinically relevant ß-lactam antibiotics. The structure-activity relationship for the C4 phenylthio ß-lactams has not yet been completely defined. Earlier efforts in our laboratories established that the C4-phenylthio substituent is essential for antimicrobial activity, while the N1 carbamyl substituent plays a more subtle role. In this present study, we investigated the role that the stereochemistry at C4 plays in these compounds' antibacterial activity. This was achieved by synthesizing and testing the antimicrobial activity of diastereomers with a chiral carbamyl group at N1. Our findings indicate that a strict stereochemistry for the C4-phenylthio ß-lactams is not required to obtain optimal anti-Mtb and anti-M. cat activity. Furthermore, the structure-bioactivity profiles more closely relate to the electronic requirement of the phenylthiogroup. In addition, the MICs of Mtb are sensitive to growth medium composition. Select compounds showed activity against non-replicating and multi-drug resistant Mtb.

Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

Non-transpeptidase binding arylthioether ß-lactams active against Mycobacterium tuberculosis and Moraxella catarrhalis.

The prevalence of drug resistance in both clinical and community settings as a consequence of alterations of biosynthetic pathways, enzymes or cell wall architecture is a persistent threat to human health. We have designed, synthesized, and tested a novel class of non-transpeptidase, ß-lactamase resistant monocyclic ß-lactams that carry an arylthio group at C4. These thioethers exhibit inhibitory and cidal activity against serine ß-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=8) ß-lactamase producing Moraxella catarrhalis clinical isolates.

Targeting host-specific metabolic pathways-opportunities and challenges for anti-infective therapy.

Microorganisms can takeover critical metabolic pathways in host cells to fuel their replication. This interaction provides an opportunity to target host metabolic pathways, in addition to the pathogen-specific ones, in the development of antimicrobials. Host-directed therapy (HDT) is an emerging strategy of anti-infective therapy, which targets host cell metabolism utilized by facultative and obligate intracellular pathogens for entry, replication, egress or persistence of infected host cells. This review provides an overview of the host lipid metabolism and links it to the challenges in the development of HDTs for viral and bacterial infections, where pathogens are using important for the host lipid enzymes, or producing their own analogous of lecithin-cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) thus interfering with the human host's lipid metabolism.

Fragment-Based Lead Discovery Strategies in Antimicrobial Drug Discovery.

Fragment-based lead discovery (FBLD) is a powerful application for developing ligands as modulators of disease targets. This approach strategy involves identification of interactions between low-molecular weight compounds (100-300 Da) and their putative targets, often with low affinity (KD ~0.1-1 mM) interactions. The focus of this screening methodology is to optimize and streamline identification of fragments with higher ligand efficiency (LE) than typical high-throughput screening. The focus of this review is on the last half decade of fragment-based drug discovery strategies that have been used for antimicrobial drug discovery.

Utilization of Existing Human Kinase Inhibitors as Scaffolds in the Development of New Antimicrobials.

The prevalence and continuing expansion of drug resistance, both in clinical and community settings represents a major challenge for current antimicrobial therapy. The different approaches for addressing this challenge include (1) identification of novel antibacterials by repurposing of existing drugs originally that historically target host proteins; and (2) effect target switching through modification of existing antimicrobials. The focus of this manuscript is on these drug discovery strategies, with utility for development of new antimicrobials with different modes of action.

The Role of the Insulin/Glucose Ratio in the Regulation of Pathogen Biofilm Formation.

During the management of patients in acute trauma the resulting transient hyperglycemia is treated by administration of insulin. Since the effect of insulin, a quorum sensing compound, together with glucose affects biofilm formation in a concentration-specific manner, we hypothesize that the insulin/glucose ratio over the physiologic range modulates biofilm formation potentially influencing the establishment of infection through biofilm formation. METHODS: A variety of Gram-positive and Gram-negative bacteria were grown in peptone (1%) yeast nitrogen base broth overnight in 96-well plates with various concentrations of glucose and insulin. Biofilm formation was determined by the crystal violet staining procedure. Expression of insulin binding was determined by fluorescent microscopy (FITC-insulin). Controls were buffer alone, insulin alone, and glucose alone. RESULTS: Overall, maximal biofilm levels were measured at 220 mg/dL of glucose, regardless of insulin concentration (10, 100, 200 µU/mL) of the organism tested. In general, insulin with glucose over the range of 160-180 mg/dL exhibited a pattern of biofilm suppression. However, either above or below this range, the presence of insulin in combination with glucose significantly modulated (increase or decrease) biofilm formation in a microbe-specific pattern. This modulation appears for some organisms to be reflective of the glucose-regulated intrinsic expression of bacterial insulin receptor expression. CONCLUSION: Insulin at physiologic levels (normal and hyperinsulinemic) in combination with glucose can affect biofilm formation in a concentration-specific and microbe-specific manner. These findings may provide insight into the importance of co-regulation of the insulin/glucose ratio in patient management.

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability.

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

Impact of host factors on susceptibility to antifungal agents.

An obstacle to drug development, particularly in this era of multiple drug resistance, is the under-appreciation for the role the host environment plays in microbial response to drugs. With the rise in fungal infection and drug resistance, particularly in individuals with co-morbidities, the influence serum and its components have on antimicrobial susceptibility requires assessment. This study examined the impact of physiologically relevant glucose and insulin levels in the presence and absence of 50 % human plasma on MICs for clinical isolates of Candida lusitaniae, Candida parapsilosis, Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei and Cryptococcus neoformans. The addition of insulin or glucose at physiologic levels in RPMI medium alone altered the MIC in either a positive or negative fashion, depending on the organisms and drug tested, with C. glabrata most significantly altered with a 40, >32- and 46-fold increase in MIC for amphotericin B, itraconazole and miconazole, respectively. The addition of candida-antibody negative plasma also affected MIC, with the addition of glucose and insulin having a tandem effect on MIC. These findings indicate that phenotypic resistance of Candida and Cryptococcus can vary depending on the presence of insulin with glucose and plasma. This modulation of resistance may help explain treatment failures in the diabetic population and facilitate the development of stable drug-resistant strains. Furthermore, these findings indicate the need for a precision approach in the choice of drug treatment and drug development.

C4-alkylthiols with activity against Moraxella catarrhalis and Mycobacterium tuberculosis.

Antimicrobial resistance represents a global threat to healthcare. The ability to adequately treat infectious diseases is increasingly under siege due to the emergence of drug-resistant microorganisms. New approaches to drug development are especially needed to target organisms that exhibit broad antibiotic resistance due to expression of ß-lactamases which is the most common mechanism by which bacteria become resistant to ß-lactam antibiotics. We designed and synthesized 20 novel monocyclic ß-lactams with alkyl- and aryl-thio moieties at C4, and subsequently tested these for antibacterial activity. These compounds demonstrated intrinsic activity against serine ß-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=6) ß-lactamase producing Moraxella catarrhalis clinical isolates.

Addressing Antimicrobial Resistance through New Medicinal and Synthetic Chemistry Strategies.

Over the past century, a multitude of derivatives of structural scaffolds with established antimicrobial potential have been prepared and tested, and a variety of new scaffolds have emerged. The effectiveness of antibiotics, however, is in sharp decline because of the emergence of drug-resistant microorganisms. The prevalence of drug resistance, both in clinical and community settings, is a consequence of bacterial ingenuity in altering pathways and/or cell morphology, making it a persistent threat to human health. The fundamental ability of pathogens to survive in a multitude of habitats can be triggered by recognition of chemical signals that warn organisms of exposure to a potentially harmful environment. Host immune defenses, including reactive oxygen intermediates and antibacterial substances, are among the multitude of chemical signals that can subsequently trigger expression of phenotypes better adapted for survival in that hostile environment. Thus, resistance development appears to be unavoidable, which leads to the conclusion that developing an alternative perspective for treatment options is vital. This review will discuss emerging medicinal chemistry approaches for addressing the global multidrug resistance in the 21st century.

Possible role of sarA in dehydroepiandrosterone-mediated increase in Staphylococcus aureus resistance to vancomycin.

BACKGROUND: Dehydroepiandrosterone (DHEA), a steroid present throughout life, can induce an increase in resistance to vancomycin in methicillin-sensitive and methicillin-resistant clinical isolates of Staphylococcus aureus. METHODS: The in vitro effect of DHEA on vancomycin killing of S. aureus with mutations in sarA and/or agr was determined by standard microtiter protocols and time to kill determinations. RESULTS: Of the isolates tested, the strain with a deletion in sarAderived from a DHEA- responsive parent was not protected from vancomycin killing by DHEA. However, DHEA significantly (p < 0.01) slowed the rate of vancomycin killing of sarA-. CONCLUSION: These data indicate that sarA may play a role in DHEA-mediated protection from vancomycin killing of S. aureus.

Chemical communication--do we have a quorum?

There are two types of bacterial communication systems, those in which the signal produced by bacteria is directed only at other organisms, and those where the signal is detected by others and self. The latter is involved in adaptation to the environment. The adaptation signals are autoinducers, the response is population density-dependent and has been termed "quorum sensing". Our current knowledge of bacterial signaling systems indicates that Gram positive bacteria use small peptides for both types of signaling, whereas Gram negative organisms use homoserine lactones as autoinducers. Gram- negative bacteria internalize the signals which act upon an intracellular receptor. Gram-positive bacteria use the signals as ligands for an extracellular receptor of a two-component signaling system. Inhibitors of quorum sensing compounds for both Gram positive and Gram negative bacteria are being explored. Signal inhibitors could be potentially effective in impeding biofilm formation, which might prolong the utility of the currently available antibiotics in this era of antibiotic resistance. In this review, we will explore both bacteria-host and bacteria-bacteria communication systems, with an emphasis on inhibitors of these systems both natural and synthetic.

Lactones: generic inhibitors of enzymes?

The ability to affect eukaryotic and prokaryotic cellular growth, signaling and differentiation is a continuing focus in the pharmaceutical industry. The fundamental ability to affect these cellular processes is inherent in lactones. Lactones, which are ubiquitous in nature, reflect a broad phylogenetic diversity indicative of their ability to act as simple alkylating compounds, with their in situ activities falling into one of two categories, i.e., protect or conquer. Medically, their utility as pharmaceutical agents range from that of antimicrobial to anti-neoplastic agent depending on the functional groups attached.

Assessment of the contributions of Na+ channel inhibition and general peripheral action in cocaine-induced conditioned taste aversion.

While the rewarding properties of cocaine appear to be mediated by its blockade of central monoamine uptake, the mechanisms and sites of action for cocaine's aversive effects have yet to be determined. Using the conditioned taste aversion (CTA) preparation, the present study examined the role of Na(+) channel blockade in cocaine's aversive effects by comparing cocaine to the local anesthetic procaine at three doses (18, 32 and 50 mg/kg). Furthermore, the role of cocaine's peripheral actions in its aversive effects was examined by comparing cocaine to the quaternary analog cocaine methiodide (equimolar to the three doses of cocaine) in establishing CTAs. Procaine and cocaine methiodide each dose-dependently suppressed saccharin consumption, indicating that the aversive effects of cocaine are, in part, mediated by its inhibition of Na(+) channels and via its activity in the PNS. However, the fact that the aversions induced by procaine and cocaine methiodide were weaker than those induced by cocaine at each dose tested suggests other factors are involved in its aversive effects. Possible reasons for the weaker aversions induced by procaine and cocaine methiodide relative to cocaine were discussed.

The relationship between inhibitors of eukaryotic and prokaryotic serine proteases.

The ability to inhibit serine proteases is a major focus in the pharmaceutical industry. Serine proteases of medical importance range in phylogenetic diversity from the metallo-proteases, which play a role in pulmonary hypertension, and destruction of the lung parenchyma in emphysema, to those proteases (beta-lactamases), which play a role in the resistance of bacteria to beta-lactam antibiotics. In both the mammalian and microbial systems, the development of serine protease inhibitors has been a focal strategy spurring investigations in the area of serine protease dependent prodrugs that incorporate a bactericidal moiety as well as other classes of metalloprotease inhibitors.

Molecular Targets of ß-Lactam-Based Antimicrobials: Beyond the Usual Suspects.

The common practice in antibacterial drug development has been to rapidly make an attempt to find ever-more stable and broad-spectrum variants for a particular antibiotic, once a drug resistance for that antibiotic is detected. We are now facing bacterial resistance toward our clinically relevant antibiotics of such a magnitude that the conversation for antimicrobial drug development ought to include effective new antibiotics with alternative mechanisms of action. The electrophilic ß-lactam ring is amenable for the inhibition of different enzyme classes by a suitable decoration of the core scaffold. Monocyclic ß-lactams lacking an ionizable group at the lactam nitrogen exhibit target preferences toward bacterial enzymes important for resistance and virulence. The present review intends to draw attention to the versatility of the ß-lactams as antimicrobials with "unusual" molecular targets.

Determination of the cation-chelating potential of C-methylthiolated beta-lactams and their sulfones by electrospray ionization mass spectrometry.

The chelation potential of highly lipophilic C-dimethylthiolated monocyclic beta-lactams was examined using electrospray ionization mass spectrometry (ESI-MS). The metal salts NaCl, KCl, CaCl2, ZnCl2, Cu(NO3)2, CdSO4, MnCl2, and Mg(NO3)2 were used for the analysis. The K+ adducts of the compounds studied were more responsive in ESI analysis, compared to their Na+ adducts, regardless of the oxidation state of the sulfur (in the methylthio or the sulfone groups) and the type of the group adjacent to the lactam carbonyl. Opening of the beta-lactam ring, leading to formation of a chargeable N-atom, had little to no effect on the K+ adduct formation. Interactions of the methylthio group with the divalent zinc ion were also observed.

Antimicrobial properties of organosulfur anti-infectives: a review of patent literature 1999-2005.

The number of organosulfur compounds being patented has been growing. A wide variety of these organosulfur compounds, whether naturally occurring or synthetic, exhibit antibacterial properties. Mechanistically, organosulfur groups can act as metal chelators, powerful nucleophiles or electrophiles depending on the local environment in which a given reaction occurs. In this review of the patent literature from 1999-2005, the reliance of these compounds on the reaction of the sulfur moiety with its biological target(s) will be discussed with regards to activity, specificity, and antimicrobial spectrum.

N-thiolated beta-lactams: a new family of anti-Bacillus agents.

This report describes the evaluation of N-thiolated beta-lactam antibiotics as potential anti-Bacillus agents. N-Thiolated beta-lactams are a new family of antibacterials that previously have been found to selectively inhibit the growth of Staphylococcus bacteria over many other genera of microbes. From the data presented herein, these lactams similarly inhibit a variety of Bacillus species, including Bacillus anthracis. The preliminary structure-activity studies suggest that there is a need to balance the lipophilic character of the C3/C4 groups in order to obtain optimal anti-Bacillus activity. Elongation or extensive branching of the organothio substituent diminishes antibacterial effects, with the sec-butylthio derivative providing the strongest activity.