Exploring the Structural Attributes of Yoda1 for the Development of New-Generation Piezo1 Agonist Yaddle1 as a Vaccine Adjuvant Targeting Optimal T Cell Activation.

Piezo1, a mechano-activated ion channel, has wide-ranging physiological and therapeutic implications, with the ongoing development of specific agonists unveiling cellular responses to mechanical stimuli. In our study, we systematically analyzed the chemical subunits in Piezo1 protein agonist Yoda1 to comprehend the structure-activity relationship and push forward next-generation agonist development. Preliminary screening assays for Piezo1 agonism were performed using the Piezo1-mCherry-transfected HEK293A cell line, keeping Yoda1 as a positive control. We introduce a novel Piezo1 agonist Yaddle1 (34, 0.40 µM), featuring a trifluoromethyl group, with further exploration through in vitro studies and density functional theory calculations, emphasizing its tetrel interactions, to act as an ambidextrous wedge between the domains of Piezo1. In contrast to the poor solubility of the established agonist Yoda1, our results showed that the kinetic solubility of Yaddle1 (26.72 ± 1.8 µM at pH 7.4) is 10-fold better than that of Yoda1 (1.22 ± 0.11 µM at pH 7.4). Yaddle1 (34) induces Ca2+ influx in human CD4+ T cell, suggesting its potential as a vaccine adjuvant for enhanced T cell activation.

IUPHAR themed review: Opioid efficacy, bias, and selectivity.

Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs, antidepressants and SSRI's. Within the opioid family mu and kappa opioid receptor are commonly targeted. However, activation of the mu opioid receptor has side effects of constipation, tolerance, dependence, euphoria, and respiratory depression; activation of the kappa opioid receptor leads to dysphoria and sedation. The side effects of mu opioid receptor activation have led to mu receptor drugs being widely abused with great overdose risk. For these reasons, newer safer opioid analgesics are in high demand. For many years a focus within the opioid field was finding drugs that activated the G protein pathway at mu opioid receptor, without activating the ß-arrestin pathway, known as biased agonism. Recent advances have shown that this may not be the way forward to develop safer analgesics at mu opioid receptor, though there is still some promise at the kappa opioid receptor. Here we discuss recent novel approaches to develop safer opioid drugs including efficacy vs bias and fine-tuning receptor activation by targeting sub-pockets in the orthosteric site, we explore recent works on the structural basis of bias, and we put forward the suggestion that Gα subtype selectivity may be an exciting new area of interest.

Development of selective class I protein arginine methyltransferase inhibitors through fragment-based drug design approach.

Protein arginine methyltransferases (PRMTs) catalyze the methylation of the terminal nitrogen atoms of the guanidino group of arginine of protein substrates. The aberrant expression of these methyltransferases is linked to various diseases, making them promising therapeutic targets. Currently, PRMT inhibitors are at different stages of clinical development, which validated their significance as drug targets. Structural Genomics Consortium (SGC) has reported several small fragment inhibitors as Class I PRMT inhibitors, which can be the starting point for rational drug development. Herein, we report the successful application of a fragment-based approach toward the discovery of selective Class I PRMT inhibitors. Structure-based ligand optimization was performed by strategic incorporation of fragment hits on the drug-like quinazoline core and subsequent fragment growth in the desired orientation towards identified hydrophobic shelf. A clear SAR was established, and the lead compounds 55 and 56 displayed potent inhibition of Class I PRMTs with IC50 values of 92 nM and 37 nM against PRMT4. We report the systematic development of potent Class I PRMT inhibitors with good potency and about 100-fold selectivity when tested against a panel of 31 human DNA, RNA, and protein lysine and arginine methyltransferases. These improved small molecules will provide new options for the development of novel potent and selective PRMT4 inhibitors.

Mitigating hERG Liability of Toll-Like Receptor 9 and 7 Antagonists through Structure-Based Design.

hERG is considered to be a primary anti-target in the drug development process, as the K+ channel encoded by hERG plays an important role in cardiac re-polarization. It is desirable to address the hERG safety liability during early-stage development to avoid the expenses of validating leads that will eventually fail at a later stage. We have previously reported the development of highly potent quinazoline-based TLR7 and TLR9 antagonists for possible application against autoimmune disease. Initial experimental hERG assessment showed that most of the lead TLR7 and TLR9 antagonists suffer from hERG liability rendering them ineffective for further development. The present study herein describes a coordinated strategy to integrate the understanding from structure-based protein-ligand interaction to develop non- hERG binders with IC50 >30 µM with retention of TLR7/9 antagonism through a single point change in the scaffold. This structure-guided strategy can serve as a prototype for abolishing hERG liability during lead optimization.

Modified Akuamma Alkaloids with Increased Potency at the Mu-opioid Receptor.

Akuammine (1) and pseudoakuammigine (2) are indole alkaloids found in the seeds of the akuamma tree (Picralima nitida). Both alkaloids are weak agonists of the mu opioid receptor (µOR); however, they produce minimal effects in animal models of antinociception. To probe the interactions of 1 and 2 at the opioid receptors, we have prepared a collection of 22 semisynthetic derivatives. Evaluation of this collection at the µOR and kappa opioid receptor (κOR) revealed structural-activity relationship trends and derivatives with improved potency at the µOR. Most notably, the introduction of a phenethyl moiety to the N1 of 2 produces a 70-fold increase in potency and a 7-fold increase in selectivity for the µOR. The in vitro potency of this compound resulted in increased efficacy in the tail-flick and hot-plate assays of antinociception. The improved potency of these derivatives highlights the promise of exploring natural product scaffolds to probe the opioid receptors.

Opioid signaling and design of analgesics.

Clinical treatment of acute to severe pain relies on the use of opioids. While their potency is significant, there are considerable side effects that can negatively affect patients. Their rise in usage has correlated with the current opioid epidemic in the United States, which has led to more than 70,000 deaths per year (Volkow and Blanco, 2021). Opioid-related drug development aims to make target compounds that show strong potency but with diminished side effects. Research into pharmaceuticals that could act as potential alternatives to current pains medications has relied on mechanistic insights of opioid receptors, a class of G-protein coupled receptors (GPCRs), and biased agonism, a common phenomenon among pharmaceutical compounds where downstream effects can be altered at the same receptor via different agonists. Opioids function typically by binding to an active site on the extracellular portion of opioid receptors. Once activated, the opioid receptor initiates a G-protein signaling pathway and/or the ß-arrestin2 pathway. The proposed concept for the development of safe analgesics around mu and kappa opioid receptor subtypes has focused on not recruiting ß-arrestin2 (biased agonism) and/or having low efficacy at the receptor (partial agonism). By altering chemical motifs on a common scaffold, chemists can take advantage of biased agonism as well as create compounds with low intrinsic efficacy for the desired treatments. This review will focus on ligands with bias profile, signaling aspects of the receptor and probe into the structural basis of receptor that leads to bias and/or partial agonism.

Synthesis and characterization of new potent TLR7 antagonists based on analysis of the binding mode using biomolecular simulations.

Aberrant activation of the endosomal Toll-like receptor 7 (TLR7) has been implicated in myriad autoimmune diseases and is an established therapeutic target in such conditions. Development of diverse TLR7 antagonists is mainly accomplished through random screening. To correlate human TLR7 (hTLR7) antagonistic activity with the structural features in different chemotypes, we derived a hypothetical binding model based on molecular docking analysis along with molecular dynamics (MD) simulations study. The binding hypothesis revealed different pockets, grooves and a central cavity where ligand-receptor interaction with specific residues through hydrophobic and hydrogen bond interactions take place, which correlate with TLR7 antagonistic activity thus paving the way for rational design using varied chemotypes. Based on the structural insight thus gained, TLR7 antagonists with quinazoline were designed to understand the effect of engagement of these pockets as well as boundaries of the chemical space associated with them. The newly synthesized most potent hTLR7 antagonist, i.e. compound 63, showed IC50 value of 1.03 ± 0.05 µM and was validated by performing primary assay in human plasmacytoid dendritic cells (pDC) (IC50pDC: 1.42 µM). The biological validation of the synthesized molecules was performed in TLR7-reporter HEK293 cells as well as in human plasmacytoid dendritic cells (pDCs). Our study provides a rational design approach thus facilitating further development of novel small molecule hTLR7 antagonists based on different chemical scaffolds.

A Chemical Switch for Transforming a Purine Agonist for Toll-like Receptor 7 to a Clinically Relevant Antagonist.

Toll-like receptor 7 (TLR7) is an established therapeutic target in myriad autoimmune disorders, but no TLR7 antagonist is available for clinical use to date. Herein, we report a purine scaffold TLR7 antagonist, first-of-its-kind to our knowledge, which was developed by rationally dissecting the structural requirements for TLR7-targeted activity for a purine scaffold. Specifically, we identified a singular chemical switch at C-2 that could make a potent purine scaffold TLR7 agonist to lose agonism and acquire antagonist activity, which could further be potentiated by the introduction of an additional basic center at C-6. We ended up developing a clinically relevant TLR7 antagonist with favorable pharmacokinetics and 70.8% oral bioavailability in mice. Moreover, the TLR7 antagonists depicted excellent selectivity against TLR8. To further validate the in vivo applicability of this novel TLR7 antagonist, we demonstrated its excellent efficacy in preventing TLR7-induced pathology in a preclinical murine model of psoriasis.

Activity-guided development of potent and selective toll-like receptor 9 antagonists.

TLR9 is one of the major innate immune receptors expressed in the endosomes of pDCs and B cells in humans. Aberrant TLR9 activation is implicated in several autoimmune and metabolic disorders as well as in sepsis, making this receptor an important therapeutic target, though specific TLR9 antagonists are yet to be available for clinical use. Here we elucidate the importance of specific physiochemical properties through substitution patterns in quinazoline scaffold to achieve potent hTLR9 inhibition at < 50 nM as well as > 600 fold selectivity against hTLR7, another closely related TLR that shares downstream signaling with TLR9 but plays distinct roles in physiology and pathology. Assays were performed using hPBMC and reporter cell lines. Favorable in vitro ADME profile, pharmacokinetics as well as validation in a clinically relevant in vivo TLR9-inhibition efficacy model in mice establish these novel TLR9-antagonists as candidate therapeutic agents in relevant clinical contexts.

Cutting Edge: Piezo1 Mechanosensors Optimize Human T Cell Activation.

TCRs recognize peptides on MHC molecules and induce downstream signaling, leading to activation and clonal expansion. In addition to the strength of the interaction of TCRs with peptides on MHC molecules, mechanical forces contribute to optimal T cell activation, as reflected by the superior efficiency of immobilized TCR-cross-linking Abs compared with soluble Abs in TCR triggering, although a dedicated mechanotransduction module is not identified. We found that the professional mechanosensor protein Piezo1 is critically involved in human T cell activation. Although a deficiency in Piezo1 attenuates downstream events on ex vivo TCR triggering, a Piezo1 agonist can obviate the need to immobilize TCR-cross-linking Abs. Piezo1-driven Ca2+ influx, leading to calpain activation and organization of cortical actin scaffold, links this mechanosensor to optimal TCR signaling. Thus, we discovered a hitherto unknown regulatory mechanism for human T cell activation and provide the first evidence, to our knowledge, for the involvement of Piezo1 mechanosensors in immune regulation.

Design and development of benzoxazole derivatives with toll-like receptor 9 antagonism.

Toll-like receptor 9 (TLR9) is a major therapeutic target for numerous inflammatory disorders. Development of small molecule inhibitors for TLR9 remains largely empirical due to lack of structural understanding of potential TLR9 antagonism by small molecules and due to the unusual topology of the ligand binding surface of the receptor. To develop a structural model for rational design of small molecule TLR9 antagonists, an enhanced homology model of human TLR9 (hTLR9) was constructed. Binding mode analysis of a series of molecules having characteristic molecular geometry, flexibility and basicity was conducted based on crystal structure of the inhibitory DNA (iDNA) bound to horse and bovine TLR9. Interaction with specific amino acid residues in four leucine rich repeat (LRR) regions of TLR9 was identified to be critical for antagonism by small molecules. The biological validation of TLR9 antagonism and its correlation with probe-receptor interactions led to a reliable model that could be used for development of novel small molecules with potent TLR9 antagonism (IC50 30-100 nM) with excellent selectivity against TLR7.

Oxidation of thiocyanate with H2O2 catalyzed by [Ru(III)(edta)(H2O)]-.

The [Ru(III)(edta)(H2O)](-) (edta(4-) = ethylenediaminetetraacetate) complex is shown to catalyze the oxidation of thiocyanate (SCN(-)) with H2O2 mimicking the action of peroxidases. The kinetics of the catalytic oxidation process was studied by using stopped-flow and rapid scan spectrophotometry as a function of [Ru(III)(edta)], [H2O2], [SCN(-)], pH (3.2-9.1) and temperature (15-30 °C). Spectral analyses and kinetic data are suggestive of a catalytic pathway in which hydrogen peroxide reacts directly with thiocyanate coordinated to the Ru(III)(edta) complex. Catalytic intermediates such as [Ru(III)(edta)(OOH)](2-) and [Ru(V)(edta)(O)](-) were found to be non-reactive in the oxidation process under the specified conditions. Formation of SO4(2-) and OCN(-) was identified as oxidation products in ESI-MS experiments. A detailed mechanism in agreement with the spectral and kinetic data is presented.