Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.     

New strategies for antibacterial drug design: targeting non-multiplying latent bacteria

During the past two decades, the number of antibacterials that has reached the marketplace each year has declined, whilst resistance to existing antibacterials has increased. New antibacterials are needed to replace those that have become less effective as a result of the emergence of a high level of resistance amongst target bacteria. Antibacterials are developed by targeting live multiplying whole bacterial cells, or essential bacterial molecules such as enzymes. Using these targets, libraries of natural, recombinant or chemically synthesised compounds are screened. Most existing antibacterials have been developed by creating novel analogues of established antibacterials, which are themselves derivatives of natural compounds. Recently, live non-multiplying bacteria have been used as targets. Bacteria in such a phase are much more tolerant to antibacterials than logarithmic phase organisms. Targeting of non-multiplying bacteria has the potential to yield new antibacterials that would shorten the duration of therapy. This would be more convenient for the patient, could reduce the incidence of adverse effects of treatment, and might reduce the emergence of antibacterial resistance. However, there is much to learn about non-multiplying bacteria, particularly the mechanisms that lie behind their profound antibacterial tolerance. New terminology has been proposed for susceptibility tests for antibacterial agents against non-multiplying bacteria, namely: the minimum stationary-cidal concentration and the minimum dormicidal concentration, which are defined as the minimum concentrations of drug that will kill stationary and dormant bacteria, respectively. The relationship between the antibiotic susceptibility of stationary and logarithmic phase bacteria is the stationary/logarithmic ratio. This terminology is suitable for both planktonic and biofilm cultures. In the future, it is likely that most antibacterial drug design will be based on existing antibacterial structures, but an increasing number of new molecular antibacterial structures may emerge from screening against multiplying and perhaps non-multiplying bacteria. The genomic approach has been disappointing so far, but it is still hoped that this will produce novel antibacterial agents.     

Emerging bacterial enzyme targets

The treatment of bacterial infections is increasingly complicated by the ability of bacteria to develop resistance to antimicrobial agents, as well as by the emergence of new pathogens with the potential for rapid global spread. Thus, there is a critical need for novel antibacterial agents and new strategies to advance the drug discovery process. In the post-genomic era, comparative genomics, functional genomics and proteomics will play important roles in identifying new enzyme targets for the discovery of novel antibacterial agents. This review will discuss bacterial enzyme targets, specifically focusing on enzymes involved in fatty acid and cell wall biosynthesis.     

新型抗菌药物研究进展

抗菌药物的研究开发处于相对低谷,而耐药菌感染治疗急需新型抗菌药物,由此所产生的矛盾需要企业、政府与科研部门协调解决。现阶段新型抗菌药物的研究与开发主要在三个方面进行:①通过结构修饰,继续对已有抗菌药物进行深入开发,获得新产品;②利用基础科学研究成果,寻找新的抗菌靶位,设计新的先导化合物,研究全新抗菌药物;③研究抗感染治疗的全新方法,如抗菌肽、噬菌体等。这些有望为耐药菌感染控制提供有效方法。     

Targeted anti bacterial therapy

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating a strong need to develop new antimicrobial agents. These new antimicrobials should possess novel modes of action and/or different cellular targets compared with the existing antibiotics. As a result, new classes of compounds designed to avoid defined resistance mechanisms are undergoing pre clinical and clinical evaluation. Microbial and phage genomic sequencing are now being used to find previously unidentified genes and their corresponding proteins. In both traditional and newly developed antibiotics, the target selectivity lies in the drug itself, in its ability to affect a mechanism that is unique to prokaryotes. As a result, a vast number of potent agents that, due to low selectivity, in addition to the pathogen also affect the eukaryote host have been excluded from use as therapeutics. Such compounds could be re-considered for clinical use if applied as part of a targeted delivery platform where the drug selectivity is replaced by target-selectivity borne by the targeting moiety. With a large number of antibodies and antibody-drug conjugates already approved or near approval as cancer therapeutics, targeted therapy is becoming increasingly attractive and additional potential targeting moieties that are non-antibody based, such as peptides, non-antibody ligand-binding proteins and even carbohydrates are receiving increasing attention. Still, targeted therapy is mostly focused on cancer, with targeted anti bacterial therapies being suggested only very recently. This review will focus in the various methods of antimicrobial targeting, by systemic and local application of targeted antimicrobial substances.     

Structure‐based drug design and in vitro testing reveal new inhibitors of enoyl‐acyl carrier protein reductases

The need for new antibacterial agents is increasingly becoming of great importance as bacterial resistance to current drugs is quickly spreading. Enoyl‐acyl carrier protein reductases (FabI) are important enzymes for fatty acid biosynthesis in bacteria and other micro‐organisms. In this project, we conducted structure‐based virtual screening against the FabI enzyme, and accordingly, 37 compounds were selected for experimental testing. Interestingly, five compounds were able to demonstrate antimicrobial effect with variable inhibition activity against various strains of bacteria and fungi. Minimum inhibitory concentrations of the active compounds were determined and showed to be in low to medium micromolar range. Subsequently, enzyme inhibition assay was carried out for our five antimicrobial hits to confirm their biological target and determine their IC₅₀ values. Three of these tested compounds exhibited inhibition activity for the FabI enzyme where our best hit MN02 had an IC₅₀ value of 7.8 μM. Furthermore, MN02 is a small bisphenolic compound that is predicted to have all required features to firmly bind with the target enzyme. To sum up, hits discovered in this work can act as a good starting point for the future development of new and potent antimicrobial agents.     

Indole naphthyridinones as inhibitors of bacterial enoyl-ACP reductases FabI and FabK

Bacterial enoyl-ACP reductase (FabI) is responsible for catalyzing the final step of bacterial fatty acid biosynthesis and is an attractive target for the development of novel antibacterial agents. Previously we reported the development of FabI inhibitor 4 with narrow spectrum antimicrobial activity and in vivo efficacy against Staphylococcus aureus via intraperitoneal (ip) administration. Through iterative medicinal chemistry aided by X-ray crystal structure analysis, a new series of inhibitors has been developed with greatly increased potency against FabI-containing organisms. Several of these new inhibitors have potent antibacterial activity against multidrug resistant strains of S. aureus, and compound 30 demonstrates exceptional oral (po) in vivo efficacy in a S. aureus infection model in rats. While optimizing FabI inhibitory activity, compounds 29 and 30 were identified as having low micromolar FabK inhibitory activity, thereby increasing the antimicrobial spectrum of these compounds to include the FabK-containing pathogens Streptococcus pneumoniae and Enterococcus faecalis. The results described herein support the hypothesis that bacterial enoyl-ACP reductases are valid targets for antibacterial agents.     

Identification of borinic esters as inhibitors of bacterial cell growth and bacterial methyltransferases, CcrM and MenH

As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes.     

膜活性多肽(MAPs)的抗菌活性及其作用机制的研究进展

膜活性多肽(MAPs)是一类具有较好抗菌活性的抗菌肽。作为先天宿主防御分子,广泛的分布于细菌、植物、无脊椎动物、脊椎动物中。膜活性多肽具有抗菌肽的结构特征,肽链通常较短,带正电荷,具有两亲性的α-螺旋或β-折叠结构,通过破坏膜的通透性杀死细菌、真菌和部分肿瘤细胞。膜活性多肽在细胞膜或细胞内部存在特定的分子靶点,并因其独特的作用机制而成为一种新型的肽类抗生素。本文主要对膜活性多肽的抗菌活性及其作用机制的研究现状和发展情况做一综述。     

Novel FabH inhibitors: a patent and article literature review (2000 – 2012)

Introduction: The traditional antimicrobial chemotherapy drugs play their effects mostly via bacterial interference with in vivo amino acids, nucleotides, amino sugars and other small molecule synthesis, or interfering the biochemical processes of these small molecules to synthesize nucleic acids, peptidoglycan and other biological macromolecules. In recent years, enzymes with single function in bacterial fatty acid synthetase system have become the genome-driven novel antibacterial drug targets. Among inhibitors of these targets, FabH inhibitors are distinguished, for their target is different from that of existing antibiotics. Therefore, discovery of FabH inhibitors might be a potential orientation to overcome bacterial resistance.

Areas covered: This review summarized new patents and articles published on FabH inhibitors from 2000 to 2012.

Expert opinion: The review gives a brief understanding about the background and development in the area of FabH inhibitors that aims to solve the bacterial resistance problem. This review puts emphasis on some typical small molecules, which participate in the process of FabH inhibition. Overall, the research scopes of antibacterial agents are getting broad. Fatty acid synthase (FAS) pathway has been proved to be a promising target for the therapy. However, claim of novel antibacterial agents with more active and higher specificity is still continued.     

Synthesis and Biological Evaluation of 2‐Mercapto‐1,3‐benzothiazole Derivatives with Potential Antimicrobial Activity

The enhancement of bacterial resistance of pathogens to currently available antibiotics constitutes a serious public health threat. So, intensive efforts are underway worldwide to develop new antimicrobial agents. To identify compounds with a potent antimicrobial profile, we designed and synthesized low molecular weight 2‐mercaptobenzothiazole derivatives 2a – 2l and 3a – 3l . Both series were screened for in‐vitro antibacterial activity against the representative panel of Gram‐positive and Gram‐negative bacteria strains. The biological screening identified compounds 2e and 2l as the most active ones showing an interesting antibacterial activity with MIC values of 3.12 μg/mL against Staphylococcus aureus and 25 μg/mL against Escherichia coli, respectively. The replacement of the S‐H by the S‐Bn moiety resulted in considerable loss of the antibacterial action of the 3a – 3l series. The antibiotic action of compounds 2e and 2l was also investigated by testing their activity against some clinical isolates with different antimicrobial resistance profile. Moreover, the involvement of the NorA efflux pump in the antibacterial activity of our molecules was evaluated. Finally, in this paper, we also describe the cytotoxic activity of the most interesting compounds by MTS assay against HeLa and MRC‐5 cell lines.     

Structure-based design, synthesis, and study of potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III as potential antimicrobial agents

Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, beta-ketoacyl-ACP synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram-positive and -negative bacteria. Small molecules that inhibit FabH enzymatic activity have the potential to be candidates within a novel class of selective, nontoxic, broad-spectrum antibacterials. Using crystallographic structural information on these highly conserved active sites and structure based drug design principles, a benzoylaminobenzoic acid series of compounds was developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity against Gram-positive and selected Gram-negative organisms.     

细菌对抗菌化合物的交叉与共耐药研究

目前细菌对抗菌药物的耐药形势日渐严峻。广泛应用于医疗、农业和食品等方面的非系统药用抗菌化合物(消毒剂、防腐剂和杀虫剂等),在提供消毒保健的同时却可促使细菌产生对各类抗菌化合物的交叉或共耐药。为此,本文重点综述了细菌对抗菌药物与消毒剂、抗菌肽和重金属类化合物的交叉及共耐药,并探讨了活性氧簇(ROS)与细菌交叉或共耐药间潜在的联系,以期增进我们对细菌如何产生对抗菌药物与非药物抗菌化合物的广泛、交叉及共耐药的理解,为制定更好的抗感染治疗策略提供参考。     

Antimicrobial compounds of low molecular mass are constitutively present in insects: characterisation of beta-alanyl-tyrosine

The number of bacterial and fungal strains that have developed resistance against the classical antibiotics continues to grow. The intensified search for new antibiotic lead compounds has resulted in the discovery of numerous endogenous peptides with antimicrobial properties in plants, bacteria and animals. Their possible applications as anti-infective agents are often limited by their size, in reference to production costs and susceptibility to proteases. In this article, we report recent isolations of antimicrobial compounds from insects, with molecular masses less than 1 kDa. Experimental approaches are discussed and the first data on the antimicrobial properties of beta-alanyl-tyrosine (252 Da), one of such low molecular mass compounds isolated from the fleshfly Neobellieria bullata, are presented. We also offer evidence for the constitutive presence of antimicrobial compounds in insects of different orders, in addition to the previously identified inducible antimicrobial peptides.     

Mechanisms of antimicrobial resistance: their clinical relevance in the new millennium

Antimicrobials show selective toxicity. Suitable targets for antimicrobials to act at include the bacterial cell wall, bacterial protein and folic acid synthesis, nucleic acid metabolism in bacteria and the bacterial cell membrane. Acquired antimicrobial resistance generally can be ascribed to one of five mechanisms. These are production of drug-inactivating enzymes, modification of an existing target, acquisition of a target by-pass system, reduced cell permeability and drug removal from the cell. Introduction of a new antimicrobial into clinical practice is usually followed by the rapid emergence of resistant strains of bacteria in some species that were initially susceptible. This has reduced the long-term therapeutic value of many antimicrobials. It used to be thought that antibacterial resistance was mainly a hospital problem but now it is also a major problem in the community. Organisms in which resistance is a particular problem in the community include members of the Enterobacteriaceae, including Salmonella spp. and Shigella spp., Mycobacterium tuberculosis, Streptococcus pneumoniae, Haemophilus influenzae and Neisseria gonorrhoeae. Multi-resistant Gram-negative rods, methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci are major causes of concern in the hospital setting. Prevalence of antibacterial resistance depends both on acquisition and spread. Decreasing inappropriate usage of antimicrobials should lessen the rate of acquisition, and spread can be minimised by sensible infection control measures.     

细胞分裂蛋白FtsZ抑制剂的研究进展

细菌耐药性问题日益严重,越来越多的致病菌对现有抗生素产生了耐药性.开发新靶点的抗生素迫在眉睫.FtsZ 是介导细菌细胞分裂的关键蛋白,由于与人类微管蛋白序列的差异,有可能设计选择性作用于细菌FtsZ而不干扰宿主细胞的抑制剂,FtsZ蛋白有希望成为抗菌药物研究的新靶点.本文从人类微管蛋白抑制剂、GTP类似物、天然产物和化合物库广筛等方面综述了以FtsZ为靶点的抗菌药物研究的最新进展.     

Are outer-membrane targets the solution for MDR Gram-negative bacteria?

The outer membrane (OM) of Gram-negative bacteria confers a significant barrier to many antibacterial agents targeting periplasmic and cytosolic functions. ‘Synergist’ approaches to disrupt the OM have been hampered by poor specificity and accompanying toxicities. The OM contains proteins required for optimal growth and pathogenesis, including lipopolysaccharide (LPS) and capsular polysaccharide (CPS) transport, porins for uptake of macromolecules, and transporters for essential elements (such as iron). Does the external proximity of these proteins offer an enhanced potential to identify effective therapies? Here, we review recent experiences in exploiting Gram-negative OM proteins (OMPs) to address the calamity of exploding antimicrobial resistance.Teaser: Multidrug-resistant (MDR) Gram-negative bacteria are a growing crisis. Few new antimicrobial chemotypes or targets have been identified after decades of screening. Are OMP targets a solution to MDR Gram-negative bacteria?     

Medicinal plants for the prevention and treatment of bacterial infections

Infectious diseases are a significant cause of morbidity and mortality worldwide, accounting for approximately 50% of all deaths in tropical countries and as much as 20% of deaths in the Americas. Despite the significant progress made in microbiology and the control of microorganisms, sporadic incidents of epidemics due to drug resistant microorganisms and hitherto unknown disease-causing microbes pose an enormous threat to public health. These negative health trends call for a global initiative for the development of new strategies for the prevention and treatment of infectious disease. For over 100 years chemical compounds isolated from medicinal plants have served as the models for many clinically proven drugs, and are now being re-assessed as antimicrobial agents. The reasons for this renaissance include a reduction in the new antibacterial drugs in the pharmaceutical pipeline, an increase in antimicrobial resistance, and the need of treatments for new emerging pathogens. Literally thousands of plant species have been tested against hundreds of bacterial strains in vitro and many medicinal plants are active against a wide range of gram positive and gram negative bacteria. However, very few of these medicinal plant extracts have been tested in animal or human studies to determine safety and efficacy. This review focuses on the medicinal plants and bacteria for which there is significant published in vitro, in vivo and clinical data available. The examples provided in this review indicate that medicinal plants offer significant potential for the development of novel antibacterial therapies and adjunct treatments (i.e. MDR pump inhibitors).     

Bacterial cell wall compounds as promising targets of antimicrobial agents II. Immunological and clinical aspects

The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.     

Molecular targets for design of novel inhibitors to circumvent aminoglycoside resistance

Aminoglycosides are a class of clinically important antibiotics used in the treatment of infections caused by Gram-positive and Gram-negative organisms. They are bactericidal, targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. Antibiotic resistance is a growing problem for all classes of anti-infective agents. One of the first groups of antibiotics to encounter the challenge of resistance was the aminoglycoside -aminocyclitol family. Initially, the resistance that emerged in organisms such as Mycobacterium tuberculosis was restricted to modification of the antibiotic targets, which we now know to be the bacterial ribosomal rRNA and proteins. As new aminoglycosides came to the clinic, however, the prevalence of chemical modification mechanisms of resistance became dominant. Enzymatic modification of aminoglycosides through kinases (O-phosphotransferases, APHs), O-adenyltransferases (ANTs) and N-acetyltransferases (AACs) has emerged in virtually all clinically relevant bacteria of both Gram-positive and Gram-negative origin. Although their clinical use has been extensive, their toxicity and the prevalence of resistance in clinical strains have prompted the pharmaceutical industry to look for alternatives. Whereas the search for novel targets for antibiotics from the genomic information is ongoing, no antibacterial agent based on these efforts has so far entered clinical trials. Meanwhile, structural knowledge of the ribosome, the target for aminoglycosides, has invigorated the field of antibiotic development. It is expected that knowledge of the binding interactions of aminoglycosides and the ribosome would lead to concepts in drug design that would take us away from the parental structures of aminoglycosides in the direction of different structural classes that bind to the same ribosomal target sites as aminoglycosides. The challenge to ensure the continued use of these highly potent antibacterial agents will require the effective management of resistance at several levels. One potential mechanism of circumventing resistance is the development of inhibitors of modification enzymes, a methodology that is now well established in the beta-lactam field. This approach requires knowledge of resistance at the molecular and atomic levels for the rational design of inhibitory molecules. The understanding of the molecular basis for aminoglycoside resistance modification has been greatly enhanced by the recent availability of representative 3D-structures from the three classes of modifying enzymes: kinases, acetyltransferases and adenyltransferases. The challenge is now to firmly establish the mechanisms of enzyme action and to use this information to prepare effective and potent inhibitors that will reverse antibiotic resistance. In this review, we discuss the molecular mechanisms of resistance of aminoglycosides specifically on aminoglycoside-modifying enzymes and newly developed strategies to circumvent resistance including antisense technology, which is an example of new strategy to deal with antibiotic resistance.