Molecular insights into the dynamic modulation of bacterial ClpP function and oligomerization by peptidomimetic boronate compounds.

Bacterial caseinolytic protease P subunit (ClpP) is important and vital for cell survival and infectivity. Recent publications describe and discuss the complex structure-function relationship of ClpP and its processive activity mediated by 14 catalytic sites. Even so, there are several aspects yet to be further elucidated, such as the paradoxical allosteric modulation of ClpP by peptidomimetic boronates. These compounds bind to all catalytic sites, and in specific conditions, they stimulate a dysregulated degradation of peptides and globular proteins, instead of inhibiting the enzymatic activity, as expected for serine proteases in general. Aiming to explore and explain this paradoxical effect, we solved and refined the crystal structure of native ClpP from Staphylococcus epidermidis (Se), an opportunistic pathogen involved in nosocomial infections, as well as ClpP in complex with ixazomib at 1.90 Å and 2.33 Å resolution, respectively. The interpretation of the crystal structures, in combination with complementary biochemical and biophysical data, shed light on how ixazomib affects the ClpP conformational state and activity. Moreover, SEC-SAXS and DLS measurements show, for the first time, that a peptidomimetic boronate compound also induces the assembly of the tetradecameric structure from isolated homomeric heptameric rings of a gram-positive organism.

Virtual Screening for Biomimetic Anti-Cancer Peptides from Cordyceps militaris Putative Pepsinized Peptidome and Validation on Colon Cancer Cell Line.

Colorectal cancer is one of the leading causes of cancer-related death in Thailand and many other countries. The standard practice for curing this cancer is surgery with an adjuvant chemotherapy treatment. However, the unfavorable side effects of chemotherapeutic drugs are undeniable. Recently, protein hydrolysates and anticancer peptides have become popular alternative options for colon cancer treatment. Therefore, we aimed to screen and select the anticancer peptide candidates from the in silico pepsin hydrolysate of a Cordyceps militaris (CM) proteome using machine-learning-based prediction servers for anticancer prediction, i.e., AntiCP, iACP, and MLACP. The selected CM-anticancer peptide candidates could be an alternative treatment or co-treatment agent for colorectal cancer, reducing the use of chemotherapeutic drugs. To ensure the anticancer properties, an in vitro assay was performed with "CM-biomimetic peptides" on the non-metastatic colon cancer cell line (HT-29). According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results from peptide candidate treatments at 0-400 µM, the IC50 doses of the CM-biomimetic peptide with no toxic and cancer-cell-penetrating ability, original C. militaris biomimetic peptide (C-ori), against the HT-29 cell line were 114.9 µM at 72 hours. The effects of C-ori compared to the doxorubicin, a conventional chemotherapeutic drug for colon cancer treatment, and the combination effects of both the CM-anticancer peptide and doxorubicin were observed. The results showed that C-ori increased the overall efficiency in the combination treatment with doxorubicin. According to the acridine orange/propidium iodine (AO/PI) staining assay, C-ori can induce apoptosis in HT-29 cells significantly, confirmed by chromatin condensation, membrane blebbing, apoptotic bodies, and late apoptosis which were observed under a fluorescence microscope.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Gramicidin-S-Inspired Cyclopeptidomimetics as Potent Membrane-Active Bactericidal Agents with Therapeutic Potential.

Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using ß,γ-diamino acids (ß,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential.

The Best Peptidomimetic Strategies to Undercover Antibacterial Peptides.

Health-care systems that develop rapidly and efficiently may increase the lifespan of humans. Nevertheless, the older population is more fragile, and is at an increased risk of disease development. A concurrently growing number of surgeries and transplantations have caused antibiotics to be used much more frequently, and for much longer periods of time, which in turn increases microbial resistance. In 1945, Fleming warned against the abuse of antibiotics in his Nobel lecture: "The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant". After 70 years, we are witnessing the fulfilment of Fleming's prophecy, as more than 700,000 people die each year due to drug-resistant diseases. Naturally occurring antimicrobial peptides protect all living matter against bacteria, and now different peptidomimetic strategies to engineer innovative antibiotics are being developed to defend humans against bacterial infections.

Integrin-mediated adhesive properties of neutrophils are reduced by hyperbaric oxygen therapy in patients with chronic non-healing wound.

In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and ß2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and ß2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4ß1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in ß2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4ß1 and ß2 was significantly reduced 70 and 67%, respectively), but α4ß1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.

Probing synergistic interplay between bio-inspired peptidomimetic chitosan-copper complexes and doxorubicin.

The current investigation reports a novel and facile method for modification of low molecular weight chitosan (Cs) with guanidine moieties, aimed at enhancing its cellular interaction and thus augmenting its cellular internalization. Guadinylated chitosan-copper (Cs-Gn-Cu) chelates, based on copper-nitrogen co-ordination, were established. Characterization of chelates was conducted using 1H NMR, 13C NMR, XPS, XRD, TGA-DTA, and GPC techniques. Anticancer activity of formed chelates was confirmed against A549 cells using MTT assay. Experimental outcomes, for the first time, have provided an empirical evidence for synergistic interaction between the chelated polymer (Cs-Gn-Cu) and the established anti-cancer agent, Doxorubicin (Dox), based on analysis by the Chou Talalay method and estimation of their combination indices. ROS induction was demonstrated as the mechanism of action of the chelated polymer, which supplemented rapid destruction of cancerous cells by Dox. These findings strongly advocate the need for harnessing unexplored potential of these innovative metal polymer chelates in cases of Dox resistant lung cancer, wherein the polymeric system itself would serve as an anti-cancer agent.

COVID-19 Coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles' heel conserved region to minimize probability of escape mutations and drug resistance.

This paper continues a recent study of the spike protein sequence of the COVID-19 virus (SARS-CoV-2). It is also in part an introductory review to relevant computational techniques for tackling viral threats, using COVID-19 as an example. Q-UEL tools for facilitating access to knowledge and bioinformatics tools were again used for efficiency, but the focus in this paper is even more on the virus. Subsequence KRSFIEDLLFNKV of the S2' spike glycoprotein proteolytic cleavage site continues to appear important. Here it is shown to be recognizable in the common cold coronaviruses, avian coronaviruses and possibly as traces in the nidoviruses of reptiles and fish. Its function or functions thus seem important to the coronaviruses. It might represent SARS-CoV-2 Achilles' heel, less likely to acquire resistance by mutation, as has happened in some early SARS vaccine studies discussed in the previous paper. Preliminary conformational analysis of the receptor (ACE2) binding site of the spike protein is carried out suggesting that while it is somewhat conserved, it appears to be more variable than KRSFIEDLLFNKV. However compounds like emodin that inhibit SARS entry, apparently by binding ACE2, might also have functions at several different human protein binding sites. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 is again argued to be a convenient model pharmacophore perhaps representing an ensemble of targets, and it is noted that it occurs both in lung and alimentary tract. Perhaps it benefits the virus to block an inflammatory response by inhibiting the dehydrogenase, but a fairly complex web involves several possible targets.

Dilipid Ultrashort Tetrabasic Peptidomimetics Potentiate Novobiocin and Rifampicin Against Multidrug-Resistant Gram-Negative Bacteria.

The development of new antibacterial agents and therapeutic approaches is of high importance to address the global problem of antibiotic resistance. Although antimicrobial peptides are known to synergize with certain antibiotics, their clinical application is limited by their systemic toxicity, protease instability, and high production cost. To overcome these problems, nine dilipid ultrashort tetrabasic peptidomimetics (dUSTBPs) were prepared consisting of three basic amino acids separated by a molecular scaffold, bis(3-aminopropyl)glycine, and were ligated to two fatty acids. Several nonhemolytic dUSTBPs were shown to enhance the activity of several antibiotics against pathogenic Gram-negative bacteria. More importantly, dUSTBP 8, consisting of three l-arginine units and a dilipid of 8 carbons long, potentiated novobiocin and rifampicin consistently against multidrug-resistant (MDR) clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae. Preliminary studies suggested that dUSTBPs were likely to potentiate antibiotics through outer membrane permeabilization and/or disruption of active efflux and that dUSTBP 8 exhibited enhanced resistance to trypsin in comparison to the previously described di-C9-KKKK-NH2 antibiotic potentiator. The antibacterial activity of rifampicin and novobiocin was enhanced by dUSTBP 8 comparable to other known outer membrane permeabilizing potentiators including the gold standard polymyxin B nonapeptide. Our results indicate that ultrashort tetrabasic peptidomimetics are potent adjuvants that repurpose novobiocin and rifampicin as potent agents against priority MDR Gram-negative pathogens.

Peptidomimetic toolbox for drug discovery.

The art of transforming peptides into drug leads is still a dynamic and fertile field in medicinal chemistry and drug discovery. Peptidomimetics can respond to peptide limitations by displaying higher metabolic stability, good bioavailability and enhanced receptor affinity and selectivity. Various synthetic strategies have been developed over the years in order to modulate the conformational flexibility and the peptide character of peptidomimetic compounds. This tutorial review aims to outline useful tools towards peptidomimetic design, spanning from local modifications, global restrictions and the use of secondary structure mimetics. Selected successful examples of each approach are presented to document the relevance of peptidomimetics in drug discovery.

A Peptidomimetic Fluorescent Probe to Detect the Trypsin ß2 Subunit of the Human 20S Proteasome.

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (ß2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ-Dap(O2(Cbz))-Dap(GO1)-Dap(O2(Cbz))-Arg-ANB-NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 µM, kcat = 245 s-1, and kcat/Km = 7.61 × 107 M-1 s-1. This process was practically halted when a selective inhibitor of the ß2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10-11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.

The Activity of Small Urea-γ-AApeptides Toward Gram-Positive Bacteria.

Host Defense Peptides (HDPs) have gained considerable interest due to the omnipresent threat of bacterial infection as a serious public health concern. However, development of HDPs is impeded by several drawbacks, such as poor selectivity, susceptibility to proteolytic degradation, low-to-moderate activity and requiring complex syntheses. Herein we report a class of lipo-linear α/urea-γ-AApeptides with a hybrid backbone and low molecular weight. The heterogeneous backbone not only enhances chemodiversity, but also shows effective antimicrobial activity against Gram-positive bacteria and is capable of disrupting bacterial membranes and killing bacteria rapidly. Given their low molecular weight and ease of access via facile synthesis, they could be practical antibiotic agents.

Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes.

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.

Cationic biaryl 1,2,3-triazolyl peptidomimetic amphiphiles targeting Clostridioides (Clostridium) difficile: Synthesis, antibacterial evaluation and an in vivo C. difficile infection model.

Clostridioides (formerly Clostridium) difficile is a Gram-positive anaerobic bacterial pathogen that causes severe gastrointestinal infection in humans. The current chemotherapeutic options are vastly inadequate, expensive and limited; this results in an exorbitant medical and financial burden. New, inexpensive chemotherapeutic treatments for C. difficile infection with improved efficacy are urgently needed. A streamlined synthetic pathway was developed to allow access to 38 novel mono- and di-cationic biaryl 1,2,3-triazolyl peptidomimetics with increased synthetic efficiency, aqueous solubility and enhanced antibacterial efficacy. The monocationic arginine derivative 28 was identified as a potent, Gram-positive selective antibacterial with MIC values of 4 µg/mL against methicillin-resistant Staphylococcus aureus and 8 µg/mL against C. difficile. Furthermore, the dicationic bis-triazole analogue 50 was found to exhibit broad-spectrum activity with substantial Gram-negative efficacy against Acinetobacter baumannii (8 µg/mL), Pseudomonas aeruginosa (8 µg/mL) and Klebsiella pneumoniae (16 µg/mL); additionally, compound 50 displayed reduced haemolytic activity (<13%) in an in vitro haemolysis assay. Membrane-disruption assays were conducted on selected derivatives to confirm the membrane-active mechanism of action inherent to the synthesized amphiphilic compounds. A comparative solubility assay was developed and utilized to optimize the aqueous solubility of the compounds for in vivo studies. The biaryl peptidomimetics 28 and 67 were found to exhibit significant efficacy in an in vivo murine model of C. difficile infection by reducing the severity and slowing the onset of disease.

Disrupting the CD95-PLCγ1 interaction prevents Th17-driven inflammation.

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.

Synthesis and Preclinical Evaluation of the First Carbon-11 Labeled PET Tracers Targeting Substance P1-7.

Two potent SP1-7 peptidomimetics have been successfully radiolabeled via [11C]CO2-fixation with excellent yields, purity, and molar activity. l-[11C]SP1-7-peptidomimetic exhibited promising ex vivo biodistribution profile. Metabolite analysis showed that l-[11C]SP1-7-peptidomimetic is stable in brain and spinal cord, whereas rapid metabolic degradation occurs in rat plasma. Metabolic stability can be significantly improved by substituting l-Phe for d-Phe, preserving 70% more of intact tracer and resulting in better brain and spinal cord tracer retention. Positron emission tomography (PET) scanning confirmed moderate brain (1.5 SUV; peak at 3 min) and spinal cord (1.0 SUV; peak at 10 min) uptake for l- and d-[11C]SP1-7-peptidomimetic. A slight decrease in SUV value was observed after pretreatment with natural peptide SP1-7 in spinal cord for l-[11C]SP1-7-peptidomimetic. On the contrary, blocking using cold analogues of l- and d-[11C]tracers did not reduce the tracers' brain and spinal cord exposure. In summary, PET scanning of l- and d-[11C]SP1-7-peptidomimetics confirms rapid blood-brain barrier and blood-spinal-cord barrier penetration. Therefore, further validation of these two tracers targeting SP1-7 is needed in order to define a new PET imaging target and select its most appropriate radiopharmaceutical.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting plasmepsins - biochemical and structural insights.

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high-resolution crystal structures of PMII-KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI-10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use. DATABASE: Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.

Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Placement of Hydroxy Moiety on Pendant of Peptidomimetic Scaffold Modulates Mu and Kappa Opioid Receptor Efficacy.

In an effort to expand the structure-activity relationship (SAR) studies of a series of mixed-efficacy opioid ligands, peptidomimetics that incorporate methoxy and hydroxy groups around a benzyl or 2-methylindanyl pendant on a tetrahydroquinoline (THQ) core of the peptidomimetics were evaluated. Compounds containing a methoxy or hydroxy moiety in the o- or m-positions increased binding affinity to the kappa opioid receptor (KOR), whereas compounds containing methoxy or hydroxy groups in the p-position decreased KOR affinity and reduced or eliminated efficacy at the mu opioid receptor (MOR). The results from a substituted 2-methylindanyl series aligned with the findings from the substituted benzyl series. Our studies culminated in the development of 8c, a mixed-efficacy MOR agonist/KOR agonist with subnanomolar binding affinity for both MOR and KOR.

Simplified AIP-II Peptidomimetics Are Potent Inhibitors of Staphylococcus aureus AgrC Quorum Sensing Receptors.

The bacterial pathogen Staphylococcus aureus controls many aspects of virulence by using the accessory gene regulator (agr) quorum sensing (QS) system. The agr system is activated by a macrocyclic peptide signal known as an autoinducing peptide (AIP). We sought to develop structurally simplified mimetics of AIPs for use as chemical tools to study QS in S. aureus. Herein, we report new peptidomimetic AgrC receptor inhibitors based on a tail-truncated AIP-II peptide that have almost analogous inhibitory activities to the parent peptide. Structural comparison of one of these peptidomimetics to the parent peptide and a highly potent, all-peptide-derived, S. aureus agr inhibitor (AIP-III D4A) revealed a conserved hydrophobic motif and overall amphipathic nature. Our results suggest that the AIP scaffold is amenable to structural mimicry and minimization for the development of synthetic agr inhibitors.