The emergence of multi-drug-resistant bacteria (known as superbugs) represents one of the greatest challenges for human health and modern medicine. Due to their remarkable ability to rapidly develop resistance to currently used antibiotics, new molecular targets for bacteria and substances capable of effectively combating related infections are still being sought. Lasso (known also as lariat) peptides are an unusual subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a structurally constrained knotted fold resembling a lasso. They are synthesized by certain groups of microorganisms as a result of complex processes involving intricate structural changes leading to the formation of the lasso structure. Reproducing these processes using known peptide synthesis methods poses a major challenge for synthetic chemistry. Lasso peptides exhibit a range of bioactivities including antibacterial activity. Due to the lasso structure, the peptides are capable of binding to new molecular targets, including atypical sides of ribosomes, in relation to currently used antibiotics. Thus, creating new mechanisms that inhibit metabolic processes leading to the death of pathogenic bacteria. This feature makes lasso peptides a potential “last chance” weapon in the fight against emerging superbugs.

Vancomycin (Van) is a glycopeptide antibiotic commonly used as a last resort for treating life-threatening infections caused by multidrug-resistant bacterial strains, such as Staphylococcus aureus and Enterococcus spp. However, its effectiveness is currently limited due to the rapidly increasing number of drug-resistant clinical strains and its inherent cytotoxicity and poor penetration into cells and specific regions of the body, such as the brain. One of the most promising strategies to enhance its efficacy appears to be the covalent attachment of cell-penetrating peptides (CPPs) to the Van structure. In this study, a series of vancomycin conjugates with CPPs—such as TP10, Tat (47–57), PTD4, and Arg₉—were designed and synthesized. These conjugates were tested for antimicrobial activity against four reference strains (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa) and two clinical drug-resistant strains: methicillin-resistant S. aureus and vancomycin-resistant E. faecium. In addition, cytotoxicity tests (using a human fibroblast cell line) and blood–brain barrier (BBB) permeability tests (using a parallel artificial membrane permeability assay—PAMPA-BBB assay) were conducted for selected compounds. Our research demonstrated that conjugation of Van with CPPs, particularly with Tat (47–57), Arg₉, or TP10, significantly enhances its antimicrobial activity against Gram-positive bacteria such as S. aureus and Enterococcus spp., reduces its cytotoxicity, and improves its access to brain tissues. We conclude that these findings provide a strong foundation for the design of novel antimicrobial agents effective in treating infections caused by drug-resistant staphylococcal and enterococcal strains, while also being capable of crossing the BBB.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran—a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate–sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT–real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.

Nucleopeptides (NPs) represent synthetic polymers created by attaching nucleobases to the side chains of amino acid residues within peptides. These compounds amalgamate the characteristics of peptides and nucleic acids, showcasing a unique ability to recognize RNA structures. In this study, we present the design and synthesis of Fmoc-protected nucleobase amino acids (1,4-TzlNBAs) and a new class of NPs, where canonical nucleobases are affixed to the side chain of L-homoalanine (Hal) through a 1,4-linked-1,2,3-triazole (HalTzl). Fmoc-protected 1,4-TzlNBAs suitable for HalTzl synthesis were obtained via Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) conjugation of Fmoc-L-azidohomoalanine (Fmoc-Aha) and N1- or N9-propargylated nucleobases or their derivatives. Following this, two trinucleopeptides, HalTzl_AAA and HalTzl_AGA, and the hexanucleopeptide HalTzl_TCCCAG, designed to complement bulge and outer loop structures of TAR (trans-activation response element) RNA HIV-1, were synthesized using the classical solid-phase peptide synthesis (SPPS) protocol. The binding between HalTzls and fluorescently labeled 5′-(FAM(6))-TAR UCU and UUU mutant was characterized using circular dichroism (CD) and fluorescence spectroscopy. CD results confirmed the binding of HalTzls to TAR RNA, which was evident by a decrease in ellipticity band intensity around 265 nm during complexation. CD thermal denaturation studies indicated a relatively modest effect of complexation on the stability of TAR RNA structure. The binding of HalTzls at an equimolar ratio only marginally increased the melting temperature (T_m) of the TAR RNA structure, with an increment of less than 2 °C in most cases. Fluorescence spectroscopy revealed that HalTzl_AAA and HalTzl_AGA, complementary to UUU or UCU bulges, respectively, exhibited disparate affinities for the TAR RNA structure (with K_d ≈ 30 and 256 µM, respectively). Hexamer HalTzl_TCCCAG, binding to the outer loop of TAR_UCU, demonstrated a moderate affinity with K_d ≈ 38 µM. This study demonstrates that newly designed HalTzls effectively bind the TAR RNA structure, presenting a potential new class of RNA binders and may be a promising scaffold for the development of a new class of antiviral drugs.