Target-based discovery of antagonists of the tick (Rhipicephalus microplus) kinin receptor identifies small molecules that inhibit midgut contractions.

BACKGROUND: A GPCR (G protein-coupled receptor) target-based approach was applied to identify antagonists of the arthropod-specific tick kinin receptor. These small molecules were expected to reproduce the detrimental phenotypic effects that had been observed in Rhipicephalus microplus females when the kinin receptor was silenced by RNA interference. Rhipicephalus microplus, the southern cattle tick, cattle fever tick, or Asian blue tick, is the vector of pathogenic microorganisms causing the deadly bovine babesiosis and anaplasmosis. The widespread resistance to acaricides in tick populations worldwide emphasizes that exploring novel targets for effective tick control is imperative. RESULTS: Fifty-three structural analogs of previously identified tick kinin antagonists were screened in a 'dual-addition' calcium fluorescence assay using a CHO-K1 cell line expressing the tick kinin receptor. Seven molecules were validated as non-cytotoxic antagonists, four of which were partial (SACC-0428764, SACC-0428780, SACC-0428800, and SACC-0428803), and three were full antagonists (SACC-0428799, SACC-0428801, and SACC-0428815). Four of these antagonists (SACC-0428764, SACC-0428780, SACC-0428799, and SACC-0428815) also inhibited the tick midgut contractions induced by the myotropic kinin agonist analog 1728, verifying their antagonistic bioactivity. The small molecules were tested on recombinant human neurokinin (NK) receptors, the one most similar to the invertebrate kinin receptors. Most molecules were inhibitors of the NK1 receptor, except SACC-0412066, a previously identified tick kinin receptor antagonist, which inhibited the NK1 receptor only at the highest concentration tested (25 µm). None of the molecules inhibited the NK3 human receptor. CONCLUSION: Molecules identified through this approach could be useful probes for studying the tick kinin signaling system and midgut physiology. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification of a copper-responsive small molecule inhibitor of uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC ΔtolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli, this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens. The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells. The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a "dual-addition" approach can detect functional agonist and antagonist "hit" molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z' factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 µM final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

A random small molecule library screen identifies novel antagonists of the kinin receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae).

BACKGROUND: The southern cattle tick, Rhipicephalus microplus, is a primary vector of the deadly bovine disease babesiosis. Worldwide populations of ticks have developed resistance to acaricides, underscoring the need for novel target discovery for tick control. The arthropod-specific R. microplus kinin receptor is such a target, previously validated by silencing, which resulted in female reproductive fitness costs, including a reduced percentage of eggs hatching. RESULTS: In order to identify potent small molecules that bind and activate or inhibit the kinin receptor, a high-throughput screening (HTS) assay was developed using a CHO-K1 cell line expressing the recombinant tick kinin receptor (BMLK3 ). A total of ~20 000 molecules from a random in-house small molecule library were screened in a 'dual-addition' calcium fluorescence assay. This was followed by dose-response validation of the hit molecules identified both from HTS and an in silico screen of ~390 000 molecules. We validated 29 antagonists, 11 of them were full antagonists with IC50 values between 0.67 and 8 µmol L-1 . To explore the structure-activity relationships (SAR) of the small molecules, we tested the activities of seven analogs of the most potent identified antagonist, additionally discovering three full antagonists and four partial antagonists. These three potent antagonists (IC50 < 3.2 µmol L-1 ) were validated in vitro using the recombinant mosquito kinin receptor and showed similar antagonistic activities. In vivo, these three compounds also inhibited the mosquito hindgut contraction rate induced by a myotropic kinin agonist analog 1728. CONCLUSION: Antagonists identified in this study could become pesticide leads and are reagents for probing the kinin signaling system. © 2021 Society of Chemical Industry.

The Cattle Fever Tick, Rhipicephalus microplus, as a Model for Forward Pharmacology to Elucidate Kinin GPCR Function in the Acari.

The success of the acaricide amitraz, a ligand of the tick tyramine/octopamine receptor (a G protein-coupled receptor; GPCR), stimulated interest on arthropod-specific GPCRs as targets to control tick populations. This search advances tick physiology because little is known about the pharmacology of tick GPCRs, their endogenous ligands or their physiological functions. Here we explored the tick kinin receptor, a neuropeptide GPCR, and its ligands. Kinins are pleiotropic insect neuropeptides but their function in ticks is unknown. The endogenous tick kinins are unknown and their cDNAs have not been cloned in any species. In contrast, more than 271 insect kinin sequences are available in the DINeR database. To fill this gap, we cloned the kinin cDNA from the cattle fever tick, Rhipicephalus microplus, which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including Ixodes scapularis. All species showed an expansion of kinin paracopies. The "kinin core" (minimal active sequence) of tick kinins FX1X2WGamide is similar to those in insects. Pro was predominant at the X2 position in tick kinins. Toward accelerating the discovery of kinin function in ticks we searched for novel synthetic receptor ligands. We developed a dual-addition assay for functional screens of small molecules and/or peptidomimetics that uses a fluorescent calcium reporter. A commercial library of fourteen small molecules antagonists of mammalian neurokinin (NK) receptors was screened using this endpoint assay. One acted as full antagonist (TKSM02) with inhibitory concentration fifty (IC50) of ∼45 µM, and three were partial antagonists. A subsequent calcium bioluminescence assay tested these four antagonists through kinetic curves and confirmed TKSM02 as full antagonist and one as partial antagonist (TKSM14). Antagonists of NK receptors displayed selectivity (>10,000-fold) on the tick kinin receptor. Three peptidomimetic ligands of the mammalian NK receptors (hemokinin 1, antagonist G, and spantide I) were tested in the bioluminescence assay but none were active. Forward approaches may accelerate discovery of kinin ligands, either as reagents for tick physiological research or as lead molecules for acaricide development, and they demonstrate that selectivity is achievable between mammalian and tick neuropeptide systems.

Mycobacterium Cytidylate Kinase Appears to Be an Undruggable Target.

New and improved drugs against tuberculosis are urgently needed as multi-drug-resistant forms of the disease become more prevalent. Mycobacterium tuberculosis cytidylate kinase is an attractive target for screening due to its essentiality and different substrate specificity to the human orthologue. However, we selected the Mycobacterium smegmatis cytidylate kinase for screening because of the availability of high-resolution X-ray crystallographic data defining its structure and the high likelihood of active site structural similarity to the M. tuberculosis orthologue. We report the development and implementation of a high-throughput luciferase-based activity assay and screening of 19,920 compounds derived from small-molecule libraries and an in silico screen predicting likely inhibitors of the cytidylate kinase enzyme. Hit validation included a counterscreen for luciferase inhibitors that would result in false positives in the initial screen. Results of this counterscreen ruled out all of the putative cytidylate kinase inhibitors identified in the initial screening, leaving no compounds as candidates for drug development. Although a negative result, this study indicates that this important drug target may in fact be undruggable and serve as a warning for future investigations.

High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors.

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors.

Studies on monitoring and tracking genetic resources: an executive summary.

The principles underlying fair and equitable sharing of benefits derived from the utilization of genetic resources are set out in Article 15 of the UN Convention on Biological Diversity, which stipulate that access to genetic resources is subject to the prior informed consent of the country where such resources are located and to mutually agreed terms regarding the sharing of benefits that could be derived from such access. One issue of particular concern for provider countries is how to monitor and track genetic resources once they have left the provider country and enter into use in a variety of forms. This report was commissioned to provide a detailed review of advances in DNA sequencing technologies, as those methods apply to identification of genetic resources, and the use of globally unique persistent identifiers for persistently linking to data and other forms of digital documentation that is linked to individual genetic resources. While the report was written for an audience with a mixture of technical, legal, and policy backgrounds it is relevant to the genomics community as it is an example of downstream application of genomics information.

The value of natural products to future pharmaceutical discovery.

Natural products have provided considerable value to the pharmaceutical industry over the past half century. In particular, the therapeutic areas of infectious diseases and oncology have benefited from numerous drug classes derived from natural product sources. Unfortunately, pharmaceutical companies have significantly decreased activities in natural product discovery during the past several years. Biotechnology companies working in the fields of combinatorial biosynthesis, genetic engineering and metagenomic approaches to identify novel natural product lead molecules have had limited success. Despite what appears to be a slow death of natural product discovery research, many new and interesting molecules with biological activity have been published in the past few years. If natural product materials continue to be tested for desirable therapeutic activities, we believe that significant progress in identifying new antibiotics, oncology therapeutics and other useful medicines will be made.

Drug discovery from natural products.

Natural product compounds are the source of numerous therapeutic agents. Recent progress to discover drugs from natural product sources has resulted in compounds that are being developed to treat cancer, resistant bacteria and viruses and immunosuppressive disorders. Many of these compounds were discovered by applying recent advances in understanding the genetics of secondary metabolism in actinomycetes, exploring the marine environment and applying new screening technologies. In many instances, the discovery of a novel natural product serves as a tool to better understand targets and pathways in the disease process. This review describes recent progress in drug discovery from natural sources including several examples of compounds that inhibit novel drug targets.

Small-molecule natural products: new structures, new activities.

A wide variety of novel small-molecule natural products has recently been reported. These compounds were isolated from marine and terrestrial sources, and from a variety of animals, plants and microorganisms. With the breadth of diversity represented in these bioactive small molecules, the future of natural product drug discovery looks bright.

14'-Hydroxymytoxin B and 16-hydroxyroridin E, two new cytotoxic trichothecenes from Myrothecium roridum.

Two new trichothecenes, 14'-hydroxymytoxin B (1) and 16-hydroxyroridin E (3), were isolated from a fermentation extract of Myrothecium roridum. The structures of 1 and 3 were determined by spectral data interpretation. Both compounds showed potent cytotoxic activity against primary soft-tissue sarcoma cells.