Antisense and Functional Nucleic Acids in Rational Drug Development.

This review is focused on antisense and functional nucleic acid used for completely rational drug design and drug target assessment, aiming to reduce the time and money spent and increase the successful rate of drug development. Nucleic acids have unique properties that play two essential roles in drug development as drug targets and as drugs. Drug targets can be messenger, ribosomal, non-coding RNAs, ribozymes, riboswitches, and other RNAs. Furthermore, various antisense and functional nucleic acids can be valuable tools in drug discovery. Many mechanisms for RNA-based control of gene expression in both pro-and-eukaryotes and engineering approaches open new avenues for drug discovery with a critical role. This review discusses the design principles, applications, and prospects of antisense and functional nucleic acids in drug delivery and design. Such nucleic acids include antisense oligonucleotides, synthetic ribozymes, and siRNAs, which can be employed for rational antibacterial drug development that can be very efficient. An important feature of antisense and functional nucleic acids is the possibility of using rational design methods for drug development. This review aims to popularize these novel approaches to benefit the drug industry and patients.

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.

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.

Versatile tools of synthetic biology applied to drug discovery and production.

Although synthetic biology is an emerging research field, which has come to prominence within the last decade, it already has many practical applications. Its applications cover the areas of pharmaceutical biotechnology and drug discovery, bringing essential novel methods and strategies such as metabolic engineering, reprogramming the cell fate, drug production in genetically modified organisms, molecular glues, functional nucleic acids and genome editing. This review discusses the main avenues for synthetic biology application in pharmaceutical biotechnology. The authors believe that synthetic biology will reshape drug development and drug production to a similar extent as the advances in organic chemical synthesis in the 20th century. Therefore, synthetic biology already plays an essential role in pharmaceutical, biotechnology, which is the main focus of this review.

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.

Designing drugs that overcome antibacterial resistance: where do we stand and what should we do?

INTRODUCTION: In recent years, infections caused by multidrug-resistant bacterial pathogens have become a huge issue to public healthcare systems. Indeed, the misuse of antibiotics has led to, over the past 30 years, the emergence of a number of resistant bacterial strains including Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli and Mycobacterium tuberculosis. Unfortunately, efforts to produce new antibiotics have not been sufficient to cope with the emergence of these new antibiotic-resistant (AR) strains. AREAS COVERED: There is an urgent need to invent and employ unconventional strategies for antimicrobial drug development to tackle the rising global threats imposed by the spread of antimicrobial resistance. Herein, the authors discuss these novel design strategies and provide their expert perspective on the subject. EXPERT OPINION: To deal with the growing threat of AR, it is important to cut down the use of antibiotics to the very minimum to diminish the risk of unknown drug-resistant bacteria and increase antibacterial vaccination programs. Furthermore, it is important to develop new classes of antibiotics that can deal with multidrug-resistant bacterial pathogens.