Discovery of the TLR7/8 Antagonist MHV370 for Treatment of Systemic Autoimmune Diseases.

Toll-like receptor (TLR) 7 and TLR8 are endosomal sensors of the innate immune system that are activated by GU-rich single stranded RNA (ssRNA). Multiple genetic and functional lines of evidence link chronic activation of TLR7/8 to the pathogenesis of systemic autoimmune diseases (sAID) such as Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). This makes targeting TLR7/8-induced inflammation with small-molecule inhibitors an attractive approach for the treatment of patients suffering from systemic autoimmune diseases.

First-in-Human Study of the Safety, Pharmacokinetics, and Pharmacodynamics of MHV370, a Dual Inhibitor of Toll-Like Receptors 7 and 8, in Healthy Adults.

Background And Objective: MHV370, a dual antagonist of human Toll-like receptors (TLR) 7 and 8, suppresses cytokines and interferon-stimulated genes in vitro and in vivo, and  has demonstrated efficacy in murine models of lupus. This first-in-human study aimed to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple doses of MHV370 in healthy adults, as well as the effects of food consumption on a single dose of MHV370.

Methods: This was a phase 1, randomised, placebo-controlled study conducted in three parts.

Preclinical characterization of the Toll-like receptor 7/8 antagonist MHV370 for lupus therapy.

Genetic and in vivo evidence suggests that aberrant recognition of RNA-containing autoantigens by Toll-like receptors (TLRs) 7 and 8 drives autoimmune diseases. Here we report on the preclinical characterization of MHV370, a selective oral TLR7/8 inhibitor. In vitro, MHV370 inhibits TLR7/8-dependent production of cytokines in human and mouse cells, notably interferon-α, a clinically validated driver of autoimmune diseases.

A root cause analysis to identify the mechanistic drivers of immunogenicity against the anti-VEGF biotherapeutic brolucizumab.

Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not.

Nonhematopoietic IRAK1 drives arthritis via neutrophil chemoattractants.

IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling.

Structure-Based Optimization of a Fragment-like TLR8 Binding Screening Hit to an Efficacious TLR7/8 Antagonist.

Inappropriate activation of TLR7 and TLR8 is linked to several autoimmune diseases, such as lupus erythematosus. Here we report on the efficient structure-based optimization of the inhibition of TLR8, starting from a co-crystal structure of a small screening hit. Further optimization of the physicochemical properties for cellular potency and expansion of the structure-activity relationship for dual potency finally resulted in a highly potent TLR7/8 antagonist with demonstrated efficacy after oral dosing.

Target-Based Identification and Optimization of 5-Indazol-5-yl Pyridones as Toll-like Receptor 7 and 8 Antagonists Using a Biochemical TLR8 Antagonist Competition Assay.

Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist () with efficacy.

Discovery of potent, orally bioavailable in vivo efficacious antagonists of the TLR7/8 pathway.

Antagonism of the Toll-like receptors (TLRs) 7 and TLR8 has been hypothesized to be beneficial to patients suffering from autoimmune conditions. A phenotypic screen for small molecule antagonists of TLR7/8 was carried out in a murine P4H1 cell line. Compound 1 was identified as a hit that showed antagonistic activity on TLR7 and TLR8 but not TLR9, as shown on human peripheral blood mononuclear cells (hPBMCs).

Modulating ADME Properties by Fluorination: MK2 Inhibitors with Improved Oral Exposure.

MAP-activated protein kinase 2 (MK2) plays an important role in the regulation of innate immune response as well as in cell survival upon DNA damage. Despite its potential for the treatment of inflammation and cancer, to date no MK2 low molecular weight inhibitors have reached the clinic, mainly due to inadequate absorption, distribution, metabolism, and excretion (ADME) properties. We describe here an approach based on specifically placed fluorine within a recently described pyrrole-based MK2 inhibitor scaffold for manipulation of its physicochemical and ADME properties.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part I.

Pyrrolo[2,3-f]isoquinoline based amino acids, tetracyclic lactams and cyclic ketone analogues are described as novel MK2 inhibitors with IC(50) as low as 5nM and good selectivity profiles against a number of related kinases including ERK, p38alpha and JNKs. TNFalpha release was suppressed from human peripheral blood mononuclear cells (hPBMCs), and a representative compound inhibited LPS induced TNFalpha release in mice illustrating the potential of this series to provide orally active MK2 inhibitors.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II.

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D-E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC(50)<3 nM and inhibit the release of TNFalpha (IC(50)<0.3 microM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells.

Novel 3-aminopyrazole inhibitors of MK-2 discovered by scaffold hopping strategy.

New, selective 3-aminopyrazole based MK2-inhibitors were discovered by scaffold hopping strategy. The new derivatives proved to inhibit intracellular phosphorylation of hsp27 as well as LPS-induced TNFalpha release in cells. In addition, selected derivative 14e also inhibited LPS-induced TNFalpha release in vivo.

Pyrrolo-pyrimidones: a novel class of MK2 inhibitors with potent cellular activity.

Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel of 28 kinases.

Extracellular loops and ligand binding to a subfamily of Family A G-protein-coupled receptors.

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins which mediate their effects by coupling to G-proteins. The V(1a)R (V(1a) vasopressin receptor) is a member of a family of related GPCRs that are activated by vasopressin {AVP ([Arg(8)]vasopressin)}, OT (oxytocin) and related peptides. These receptors are members of a subfamily of Family A GPCRs called the neurohypophysial peptide hormone receptor family.

Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified.

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle.

Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression.

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling.

Pharmacological chaperone activity of SR49059 to functionally recover misfolded mutations of the vasopressin V1a receptor.

Pharmacological chaperones represent a new class of ligand with the potential to facilitate the delivery of misfolded, but still active, G-protein-coupled receptors to the cell surface. Using transfected HEK 293T cells, treatment with a nonpeptide antagonist, SR49059, dramatically increased ( approximately 60-fold) the surface expression of a misfolded, nonfunctional and intracellularly localized vasopressin V(1a) receptor (V(1a)R) mutant (D148A). This rescue of surface expression (111 +/- 7%) was almost identical to wild type assessed by confocal microscopy and quantitative enzyme-linked immunosorbent assay-based techniques.

Charged residues of the conserved DRY triplet of the vasopressin V1a receptor provide molecular determinants for cell surface delivery and internalization.

The highly conserved "Asp-Arg-Tyr" triplet in the distal region of the third transmembrane region of most G-protein-coupled receptors is implicated in their activation process and mediation of G-protein signaling. The aim of this study was to determine whether specific features at this locus are important for the vasopressin V(1a) receptor (V(1a)R) by performing site-directed mutagenesis. In transfected HEK 293T cells, mutation of Asp (D148A) resulted in a misfolded receptor that was nonfunctional, localized intracellularly, and not constitutively active.

The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling.

It is fundamentally important to define how agonist-receptor interaction differs from antagonist-receptor interaction. The V1a vasopressin receptor (V1aR) is a member of the neurohypophysial hormone subfamily of G protein-coupled receptors. Using alanine-scanning mutagenesis of the N-terminal juxtamembrane segment of the V1aR, we now establish that Glu54 (1.

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists.

Non-peptide antagonists of the oxytocin receptor (OTR) have been developed to prevent pre-term labour. The benzoxazinone-based antagonists L-371,257 and L-372,662 display pronounced species-dependent pharmacology with respect to selectivity for the OTR over the V(1a) vasopressin receptor. Examination of receptor sequences from different species identified Ala(318) in helix 7 of the human OTR as a candidate discriminator required for high affinity binding.

Agonist binding to peptide hormone receptors.

A fundamental issue in molecular pharmacology is to define how agonist-receptor interaction differs from that of antagonist-receptor interaction. The V(1a) vasopressin receptor (V(1a)R) is a member of a family of related G-protein-coupled receptors (GPCRs) that are activated by vasopressin, oxytocin (OT) and related peptides. A segment of the N-terminus that was required for agonist binding, but not antagonist binding, was identified by characterizing truncated V(1a)R constructs.

Curcumin: a new cell-permeant inhibitor of the inositol 1,4,5-trisphosphate receptor.

Curcumin (diferuoylmethane or 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6-hepatadiene-3,5-dione) is the active ingredient of the spice turmeric. Curcumin has been shown to have a number of pharmacological and therapeutic uses. This study shows that curcumin is a potent inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel (InsP3 receptor).

Agonist-specific, high-affinity binding epitopes are contributed by an arginine in the N-terminus of the human oxytocin receptor.

The effects of the peptide hormone oxytocin (OT) are mediated by the oxytocin receptor, which is a member of the G-protein-coupled receptor family. Defining differences between the binding of agonists and antagonists to the OTR, at the molecular level, is of fundamental importance to understanding OTR activation and to rational drug design. Previous reports have indicated that the N-terminus of the OTR is required for OT binding.

A single residue (arg46) located within the N-terminus of the V1a vasopressin receptor is critical for binding vasopressin but not peptide or nonpeptide antagonists.

A fundamental issue in molecular endocrinology is to define how agonist:receptor interaction differs from antagonist:receptor interaction. The vasopressin V1a receptor (V1aR) is a member of a subfamily of related G protein-coupled receptors that are activated by the hormone AVP or related peptides. The N-terminus of the V1aR has recently been shown to be critical for binding agonists but not antagonists.