Next generation Glucose-1-phosphate thymidylyltransferase (RmlA) inhibitors: An extended SAR study to direct future design.

The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by glucose-1-phosphate thymidylyltransferase (RmlA), which condenses glucose-1-phosphate (Glu-1-P) with deoxythymidine triphosphate (dTTP) to yield dTDP-d-glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor's pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.

Modeling the Kinetics of the Permeation of Antibacterial Agents into Growing Bacteria and Its Interplay with Efflux.

A mathematical model of the passive permeation of a novel solute into bacteria that explicitly accounts for intracellular dilution through growth was developed. A bacterial cell envelope permeability coefficient of approximately >10-8 cm2 · s-1 is predicted to ensure passive permeation into rapidly replicating bacterial cells. The relative importance of the permeability coefficients of the cytoplasmic and outer membranes of Gram-negative bacteria in determining the overall envelope permeability coefficient was analyzed quantitatively. A mathematical description of the balance between passive influx and active efflux was also developed and shows that bacterial expansion through growth can usually be neglected for compounds likely to be prepared in antibacterial drug discovery programs and the balance between passive inward permeation and active outwardly directed efflux predominates. A new parameter, efflux efficiency (η, where η is equal to k/P, in which k is the rate coefficient for the efflux pump and P is the permeability coefficient for the membrane across which the pump acts), is introduced, and the consequences for the efficiency of efflux pumping by a single pump, two pumps in parallel across either the cytoplasmic or the outer membrane, and two pumps in series, one across the cytoplasmic membrane and one across the outer membrane of Gram-negative bacteria, are explored. The results, showing additive efficiency for two pumps acting across a single membrane and multiplicative efficiency for two pumps acting in series across the cytoplasmic and outer membranes, can be quantitatively related to the ratios between MICs measured against pump-sufficient and pump deletion strains and agree with those of previous experimental and theoretical studies.

Avalanche for shape and feature-based virtual screening with 3D alignment.

This report introduces a new ligand-based virtual screening tool called Avalanche that incorporates both shape- and feature-based comparison with three-dimensional (3D) alignment between the query molecule and test compounds residing in a chemical database. Avalanche proceeds in two steps. The first step is an extremely rapid shape/feature based comparison which is used to narrow the focus from potentially millions or billions of candidate molecules and conformations to a more manageable number that are then passed to the second step. The second step is a detailed yet still rapid 3D alignment of the remaining candidate conformations to the query conformation. Using the 3D alignment, these remaining candidate conformations are scored, re-ranked and presented to the user as the top hits for further visualization and evaluation. To provide further insight into the method, the results from two prospective virtual screens are presented which show the ability of Avalanche to identify hits from chemical databases that would likely be missed by common substructure-based or fingerprint-based search methods. The Avalanche method is extended to enable patent landscaping, i.e., structural refinements to improve the patentability of hits for deployment in drug discovery campaigns.

High-throughput screening of small-molecules libraries identified antibacterials against clinically relevant multidrug-resistant A. baumannii and K. pneumoniae.

BACKGROUND: The current pipeline for new antibiotics fails to fully address the significant threat posed by drug-resistant Gram-negative bacteria that have been identified by the World Health Organization (WHO) as a global health priority. New antibacterials acting through novel mechanisms of action are urgently needed. We aimed to identify new chemical entities (NCEs) with activity against Klebsiella pneumoniae and Acinetobacter baumannii that could be developed into a new treatment for drug-resistant infections. METHODS: We developed a high-throughput phenotypic screen and selection cascade for generation of hit compounds active against multidrug-resistant (MDR) strains of K. pneumoniae and A. baumannii. We screened compound libraries selected from the proprietary collections of three pharmaceutical companies that had exited antibacterial drug discovery but continued to accumulate new compounds to their collection. Compounds from two out of three libraries were selected using "eNTRy rules" criteria associated with increased likelihood of intracellular accumulation in Escherichia coli. FINDINGS: We identified 72 compounds with confirmed activity against K. pneumoniae and/or drug-resistant A. baumannii. Two new chemical series with activity against XDR A. baumannii were identified meeting our criteria of potency (EC50 ≤50 µM) and absence of cytotoxicity (HepG2 CC50 ≥100 µM and red blood cell lysis HC50 ≥100 µM). The activity of close analogues of the two chemical series was also determined against A. baumannii clinical isolates. INTERPRETATION: This work provides proof of principle for the screening strategy developed to identify NCEs with antibacterial activity against multidrug-resistant critical priority pathogens such as K. pneumoniae and A. baumannii. The screening and hit selection cascade established here provide an excellent foundation for further screening of new compound libraries to identify high quality starting points for new antibacterial lead generation projects. FUNDING: BMBF and GARDP.

PROTAC antibiotics: the time is now.

INTRODUCTION: Novel antibiotics are needed to keep antibiotic resistance at bay and to improve treatment of the many drug-susceptible infections for which current therapies achieve poor cure rates. While revolutionizing human therapeutics, the concept of targeted protein degradation (TPD) by bifunctional proteolysis targeting chimeras (PROTACs) has not yet been applied to the discovery of antibiotics. A major obstacle precluding successful translation of this strategy to antibiotic development is that bacteria lack the E3 ligase-proteasome system exploited by human PROTACs to facilitate target degradation. AREAS COVERED: The authors describe the serendipitous discovery of the first monofunctional target-degrading antibiotic pyrazinamide, supporting TPD as a viable and novel approach in antibiotic discovery. They then discuss the rational design, mechanism, and activity of the first bifunctional antibacterial target degrader BacPROTAC, enabling a generalizable approach to TPD in bacteria. EXPERT OPINION: BacPROTACs demonstrate that linking a target directly to a bacterial protease complex can promote target degradation. BacPROTACs successfully bypass the 'middleman' E3 ligase, providing an entry strategy for the generation of antibacterial PROTACs. We speculate that antibacterial PROTACs will not only expand the target space but may also improve treatment by allowing dosage reduction, stronger bactericidal activity and activity against drug-tolerant 'persisters.'

Targeting FMN, TPP, SAM-I, and glmS Riboswitches with Chimeric Antisense Oligonucleotides for Completely Rational Antibacterial Drug Development.

Antimicrobial drug resistance has emerged as a significant challenge in contemporary medicine due to the proliferation of numerous bacterial strains resistant to all existing antibiotics. Meanwhile, riboswitches have emerged as promising targets for discovering antibacterial drugs. Riboswitches are regulatory elements in certain bacterial mRNAs that can bind to specific molecules and control gene expression via transcriptional termination, prevention of translation, or mRNA destabilization. By targeting riboswitches, we aim to develop innovative strategies to combat antibiotic-resistant bacteria and enhance the efficacy of antibacterial treatments. This convergence of challenges and opportunities underscores the ongoing quest to revolutionize medical approaches against evolving bacterial threats. For the first time, this innovative review describes the rational design and applications of chimeric antisense oligonucleotides as antibacterial agents targeting four riboswitches selected based on genome-wide bioinformatic analyses. The antisense oligonucleotides are coupled with the cell-penetrating oligopeptide pVEC, which penetrates Gram-positive and Gram-negative bacteria and specifically targets glmS, FMN, TPP, and SAM-I riboswitches in Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli. The average antibiotic dosage of antisense oligonucleotides that inhibits 80% of bacterial growth is around 700 nM (4.5 µg/mL). Antisense oligonucleotides do not exhibit toxicity in human cell lines at this concentration. The results demonstrate that these riboswitches are suitable targets for antibacterial drug development using antisense oligonucleotide technology. The approach is fully rational because selecting suitable riboswitch targets and designing ASOs that target them are based on predefined criteria. The approach can be used to develop narrow or broad-spectrum antibiotics against multidrug-resistant bacterial strains for a short time. The approach is easily adaptive to new resistance using targeting NGS technology.

Chemical Exploration of a Highly Selective Scaffold with Activity against Intracellular Mycobacterium tuberculosis.

We previously identified a phenylthiourea series with activity against intracellular Mycobacterium tuberculosis using a high-throughput, high-content assay. We conducted a catalog structure-activity relationship study with a collection of 35 analogs. We identified several thiourea derivatives with excellent potency against intracellular bacteria and good selectivity over eukaryotic cells. Compounds had much lower activity against extracellular bacteria, which was not increased by using cholesterol as the sole carbon source. Compounds were equally active against strains with mutations in QcrB or MmpL3, thereby excluding common, promiscuous targets as the mode of action. The phenylthiourea series represents a good starting point for further exploration to develop novel antitubercular agents. IMPORTANCE Mycobacterium tuberculosis is responsible for the highest number of deaths from a bacterial pathogen, with >1.5 million in 2020. M. tuberculosis is a sophisticated pathogen that can replicate inside immune cells. There is an urgent need for new drugs to combat M. tuberculosis and to shorten therapy from 6 to 24 months. We have identified a series of molecules that inhibit the growth of M. tuberculosis inside macrophages; we tested a number of derivatives to link structural features to biological activity. The compounds are likely to have novel mechanism of action and so could be developed as new agents for drug-resistant tuberculosis.

Bioinformatics and Genomic Analyses of the Suitability of Eight Riboswitches for Antibacterial Drug Targets.

Antibiotic resistance (AR) is an acute problem that results in prolonged and debilitating illnesses. AR mortality worldwide is growing and causes a pressing need to research novel mechanisms of action and untested target molecules. This article presents in silico analyses of eight bacterial riboswitches for their suitability for antibacterial drug targets. Most bacterial riboswitches are located in the 5'-untranslated region of messenger RNAs, act as allosteric cis-acting gene control elements, and have not been found in humans before. Sensing metabolites, the riboswitches regulate the synthesis of vital cellular metabolites in various pathogenic bacteria. The analyses performed in this article represent a complete and informative genome-wide bioinformatics analysis of the adequacy of eight riboswitches as antibacterial drug targets in different pathogenic bacteria based on four criteria. Due to the ability of the riboswitch to control biosynthetic pathways and transport proteins of essential metabolites and the presence/absence of alternative biosynthetic pathways, we classified them into four groups based on their suitability for use as antibacterial drug targets guided by our in silico analyses. We concluded that some of them are promising targets for antibacterial drug discovery, such as the PreQ1, MoCo RNA, cyclic-di-GMP I, and cyclic-di-GMP II riboswitches.

Covalent DNA Binding Is Essential for Gram-Negative Antibacterial Activity of Broad Spectrum Pyrrolobenzodiazepines.

It is urgent to find new antibiotic classes against multidrug-resistant bacteria as the rate of discovery of new classes of antibiotics has been very slow in the last 50 years. Recently, pyrrolobenzodiazepines (PBDs) with a C8-linked aliphatic-heterocycle have been identified as a new broad-spectrum antibiotic class with activity against Gram-negative bacteria. The active imine moiety of the reported lead pyrrolobenzodiazepine compounds was replaced with amide to obtain the non-DNA binding and noncytotoxic dilactam analogues to understand the structure-activity relationship further and improve the safety potential of this class. The synthesised compounds were tested against panels of multidrug-resistant Gram-positive and Gram-negative bacteria, including WHO priority pathogens. Minimum inhibitory concentrations for the dilactam analogues ranged from 4 to 32 mg/L for MDR Gram-positive bacteria, compared to 0.03 to 2 mg/L for the corresponding imine analogues. At the same time, they were found to be inactive against MDR Gram-negative bacteria, with a MIC > 32 mg/L, compared to a MIC of 0.5 to 32 mg/L for imine analogues. A molecular modelling study suggests that the lack of imine functionality also affects the interaction of PBDs with DNA gyrase. This study suggests that the presence of N10-C11 imine moiety is crucial for the broad-spectrum activity of pyrrolobenzodiazepines.

Targeting SAM-I Riboswitch Using Antisense Oligonucleotide Technology for Inhibiting the Growth of Staphylococcus aureus and Listeria monocytogenes.

With the discovery of antibiotics, a productive period of antibacterial drug innovation and application in healthcare systems and agriculture resulted in saving millions of lives. Unfortunately, the misusage of antibiotics led to the emergence of many resistant pathogenic strains. Some riboswitches have risen as promising targets for developing antibacterial drugs. Here, we describe the design and applications of the chimeric antisense oligonucleotide (ASO) as a novel antibacterial agent. The pVEC-ASO-1 consists of a cell-penetrating oligopeptide known as pVEC attached to an oligonucleotide part with modifications of the first and the second generations. This combination of modifications enables specific mRNA degradation under multiple turnover conditions via RNase H. The pVEC-ASO targets the S-adenosyl methionine (SAM)-I riboswitch found in the genome of many Gram-positive bacteria. The SAM-I riboswitch controls not only the biosynthesis but also the transport of SAM. We have established an antibiotic dosage of 700 nM (4.5 µg/mL) of pVEC-ASO that inhibits 80% of the growth of Staphylococcus aureus and Listeria monocytogenes. The pVEC-ASO-1 does not show any toxicity in the human cell line at MIC80's concentration. We have proven that the SAM-I riboswitch is a suitable target for antibacterial drug development based on ASO. The approach is rational and easily adapted to other bacterial RNA targets.

Targeting TPP Riboswitches Using Chimeric Antisense Oligonucleotide Technology for Antibacterial Drug Development.

Nowadays, the emergence and the transmission of multidrug-resistant pathogenic bacteria are a severe menace mounting a lot of pressure on the healthcare systems worldwide. Many severe outbreaks of bacterial infections have been reported worldwide in recent years. Thus, there is an immediate demand to develop antibiotics. Some riboswitches are potential targets for overcoming bacterial resistance. This paper demonstrates the bacteriostatic effect of an antisense oligonucleotide (ASO) engineered to suppress the growth of pathogenic bacteria such as Listeria monocytogenes by targeting the Thiamine Pyrophosphate (TPP) riboswitch. It does not inhibit the growth of the conditional pathogenic bacteria Escherichia coli, as it lacks the TPP riboswitch, showing the specificity of action of our ASO. It is covalently bonded with the cell-penetrating protein pVEC. We did bioinformatics analyses of the thiamine pyrophosphate riboswitch regarding its role in synthesizing the metabolite thiamine pyrophosphate, which is essential for bacteria. L. monocytogenes is intrinsically resistant to cephalosporins and usually is treated with ampicillin. A dosage of ASO has been established that inhibits 80% of bacterial growth at 700 nM (4.5 µg/mL). Thus, the TPP riboswitch is a valuable antibacterial target.

Engineering Antisense Oligonucleotides as Antibacterial Agents That Target FMN Riboswitches and Inhibit the Growth of Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli.

In the past several decades, antibiotic drug resistance has emerged as a significant challenge in modern medicine due to the rise of many bacterial pathogenic strains resistant to all known antibiotics. At the same time, riboswitches have emerged as novel targets for antibacterial drug discovery. Here for the first time, we describe the design and applications of antisense oligonucleotides as antibacterial agents that target a riboswitch. The antisense oligonucleotides are covalently coupled with two different cell-penetrating peptides, penetrating Gram-positive and Gram-negative bacterial cells. We specifically target Flavin MonoNucleotide (FMN) riboswitches in Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli that control both synthesis and import of FMN precursors. We have established an average antibiotic dosage by antisense oligonucleotides that inhibit 80% of bacterial growth at 700 nM (4.5 µg/mL). Furthermore, the antisense oligonucleotides do not exhibit toxicity in human cell lines at this concentration. The results demonstrate that riboswitches are suitable targets in antisense technology for antibacterial drug development.

Targeting glmS Ribozyme with Chimeric Antisense Oligonucleotides for Antibacterial Drug Development.

Due to the steady rise of multidrug-resistant pathogenic bacteria worldwide, it is critical to develop novel antibacterial drugs. This article presents chimeric antisense oligonucleotides that inhibit the bacterial growth of Staphylococcus aureus, one of the most frequent causes of hospital-acquired infections. The chimeric antisense oligonucleotides have a combination of first- and second-generation chemical modification. To deliver the antisense oligonucleotides into a cell, we apply a cell-penetrating oligopeptide attached to them. We have performed complete bioinformatics analyses of the glmS ribozyme present in S. aureus and its essential role in the biochemical pathway of glucosamine-6-phosphate synthesis. Besides, we have analyzed the bacteria for alternative metabolic pathways, such as the nagA gene. The first antisense oligonucleotide explicitly targets the glmS riboswitch, while the second explicitly targets the nagA mRNA. We have evaluated that combined, the antisense oligonucleotides block the synthesis of glucosamine-6-phosphate entirely and inhibit the bacterial growth of S. aureus. However, the glmS riboswitch targeting the antisense oligonucleotide is sufficient to inhibit the growth of S. aureus with a MIC80 of 5 µg/mL. The glmS ribozyme is a very suitable target for antibacterial drug development with antisense oligonucleotides.

Sanguinarine Inhibits the 2-Ketogluconate Pathway of Glucose Utilization in Pseudomonas aeruginosa.

Interfering with the ability of pathogenic bacteria to import glucose may represent a new promising antibacterial strategy, especially for the treatment of infections occurring in diabetic and other hyperglycemic patients. Such patients are particularly susceptible to infections caused by a variety of bacteria, among which opportunistic pathogens like Pseudomonas aeruginosa. In P. aeruginosa, glucose can be directly imported into the cytoplasm or after its periplasmic oxidation into gluconate and 2-ketogluconate (2-KG). We recently demonstrated that a P. aeruginosa mutant lacking the 2-KG transporter KguT is less virulent than its kguT + parental strain in an insect infection model, pointing to 2-KG branch of glucose utilization as a possible target for anti-Pseudomonas drugs. In this work, we devised an experimental protocol to find specific inhibitors of the 2-KG pathway of P. aeruginosa glucose utilization and applied it to the screening of the Prestwick Chemical Library. By exploiting mutants lacking genes involved in the transport of glucose derivatives in the primary screening and in the secondary assays, we could identify sanguinarine as an inhibitor of 2-KG utilization. We also demonstrated that sanguinarine does not prevent 2-KG formation by gluconate oxidation or its transport, suggesting that either KguD or KguK is the target of sanguinarine in P. Aeruginosa.

Genetic and Chemical Screening in Human Blood Serum Reveals Unique Antibacterial Targets and Compounds against Klebsiella pneumoniae.

Antibiotics halt the growth of bacteria by targeting core, essential physiology that is required for life on standard microbiological media. Many more biochemical and virulence processes, however, are required for bacteria to cause infection in a host. Indeed, chemical inhibitors of the latter processes are overlooked using conventional antibiotic drug discovery approaches. Here, we use human blood serum as an alternative growth medium to explore new targets and compounds. High-throughput screening of genetic and chemical libraries identified compounds targeting biological activities required by Klebsiella pneumoniae to grow in serum, such as nucleobase biosynthesis and iron acquisition, and showed that serum can chemically transform compounds to reveal cryptic antibacterial activity. One of these compounds, ruthenium red, was effective in a rat bloodstream infection model. Our data demonstrate that human serum is an effective tool to find new chemical matter to address the current antibiotic resistance crisis.

Genome-wide bioinformatics analysis of FMN, SAM-I, glmS, TPP, lysine, purine, cobalamin, and SAH riboswitches for their applications as allosteric antibacterial drug targets in human pathogenic bacteria.

Objectives: A constantly growing number of antibiotic-resistant strains of human pathogenic bacteria is an acute problem. Prolonged illnesses and increasing mortality worldwide mean that there is an urgent need to develop novel antibacterial drugs based on new targets and mechanisms of action. We present in silico analyses of bacterial riboswitches that may be suitable as antibacterial drug targets. Methods: Most bacterial riboswitches are allosteric cis-acting gene control elements located in the 5'-untranslated region of messenger RNAs. Riboswitches sense specific metabolites and regulate the synthesis of some essential cellular metabolites in many pathogenic bacteria but are not found in humans. We present a complete and comprehensive genome-wide bioinformatics analyses of the suitability of eight riboswitches as antibacterial drug targets in various pathogenic bacteria. Results: Based on our in silico analyses, we classify the riboswitches in four different groups based on their suitability to be used as antibacterial drug targets. We have estimated that FMN, SAM-I, glmS, TPP, and Lysine riboswitches are promising targets for antibacterial drug discovery. Conclusion: This research enables us to focus antibacterial drug discovery research only on those riboswitches whose inhibition will result in suppression of the growth of certain pathogenic bacteria.

Small steps to new drugs for bugs.

Governments, academics and industry are beginning to listen to the medical communities call for new anti-bacterials. This special issue brings together diverse review articles on topics from economics and pricing to new discovery methods.