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. In part A, participants received (3:1) a single ascending dose (SAD) of 1, 3, 10, 20, 40, 80, 160, 320, 640 and 1000 mg MHV370 or placebo. In part B, participants received (3:1) multiple ascending doses (MAD) of 25, 50, 100, 200 and 400 mg MHV370 twice daily (b.i.d) or placebo for 14 days. In part C, participants received an open-label single dose of 200 mg MHV370 under fasted or fed conditions. Safety, pharmacokinetic and pharmacodynamic parameters were evaluated. RESULTS: MHV370 was well tolerated, and no safety signal was observed in the study. No dose-limiting adverse events occurred across the dose range evaluated. Plasma concentrations of MHV370 increased with dose (mean [SD] maximum plasma concentrations ranged from 0.97 [0.48] to 1670 [861.0] ng/mL for SAD of 3-1000 mg, 29.5 [7.98] to 759 [325.0] ng/mL for MAD of 25-400 mg b.i.d. on day 1). The intake of food did not have a relevant impact on the pharmacokinetics of MHV370. Pharmacodynamic data indicated time- and dose-dependent inhibition of TLR7-mediated CD69 expression on B cells (100% inhibition at 24 h post-dose starting from SAD 160 mg and MAD 50 mg b.i.d.) and TLR8-mediated TNF release after ex vivo stimulation (>90% inhibition at 24 h post-dose starting from SAD 320 mg and MAD 100 mg b.i.d.). CONCLUSION: The safety, pharmacokinetic and pharmacodynamic data support the further development of MHV370 in systemic autoimmune diseases driven by the overactivation of TLR7 and TLR8.

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. Here, we describe how structure-based optimization of compound 2 resulted in the discovery of 34 (MHV370, (S)-N-(4-((5-(1,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methyl)bicyclo[2.2.2]octan-1-yl)morpholine-3-carboxamide). Its in vivo activity allows for further profiling toward clinical trials in patients with autoimmune disorders, and a Phase 2 proof of concept study of MHV370 has been initiated, testing its safety and efficacy in patients with Sjögren's syndrome and mixed connective tissue disease.

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. Moreover, MHV370 abrogates B cell, plasmacytoid dendritic cell, monocyte, and neutrophil responses downstream of TLR7/8. In vivo, prophylactic or therapeutic administration of MHV370 blocks secretion of TLR7 responses, including cytokine secretion, B cell activation, and gene expression of, e.g., interferon-stimulated genes. In the NZB/W F1 mouse model of lupus, MHV370 halts disease. Unlike hydroxychloroquine, MHV370 potently blocks interferon responses triggered by specific immune complexes from systemic lupus erythematosus patient sera, suggesting differentiation from clinical standard of care. These data support advancement of MHV370 to an ongoing phase 2 clinical trial.

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. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.

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. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.

Structure-Based Optimization of a Fragment-like TLR8 Binding Screening Hit to an In Vivo 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 in vivo 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 3 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 (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.

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). It was functionally cross reactive with mouse TLR7 but lacked oral exposure and had only modest potency. Chemical optimization resulted in 2, which showed in vivo efficacy following intraperitoneal administration. Further optimization resulted in 8 which had excellent in vitro activity, exposure and in vivo activity. Additional work to improve physical properties resulted in 15, an advanced lead that had favorable in vitro and exposure properties. It was further demonstrated that activity of the series tracked with binding to the extracellular domain of TLR7 implicating that the target of this series are endosomal TLRs rather than downstream signaling pathways.

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. While preserving target potency, the novel fluoro-derivatives showed greatly improved permeability as well as enhanced solubility and reduced in vivo clearance leading to significantly increased oral exposure.

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. The spirocyclopropane analogues are less potent against MK2 (IC(50)=0.05-0.23 microM), less potent in cells (IC(50)<1.1 microM), but show good oral absorption. Compound 13E (100mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.

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.

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. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V(1a) vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe(189), Trp(206), Phe(209), and Tyr(218) are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp(206) and Phe(209) project into the binding crevice. Indeed, Phe(209) is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe(189) and Tyr(218), located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

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. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

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. Recovery was not specific to D148A, since other surface-impaired mutations, D148N and D148E, and wild type were also increased following SR49059 exposure. However, surface delivery was specific to SR49059, since V(1a)R-selective peptide ligands or unrelated ligands were unable to mimic this action, suggesting that SR49059 acts intracellularly. SR49059-mediated surface rescue was time-, mutant-, and concentration-dependent but not directly related to its binding affinity. Maximal recovery was achieved following 12 h of treatment and did not involve de novo receptor synthesis or a consequence of preventing endogenous constitutive activity and/or internalization. Once at the surface, all mutants displayed enhanced signaling ability, and D148A was able to undergo agonist-mediated internalization. SR49059 was not effectively removed from the receptor, since signaling (EC(50)) of both wild type and D148A was reduced approximately 40-fold. This is the first report of a pharmacological chaperone ligand to act on misfolded mutant V(1a) Rs. This work provides an excellent model to understand the mechanistic action of an important new class of drug that may have potential in the treatment of diseases caused by inherited mutations.

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. Nonconservative (D148R) substitution was not expressed, whereas asparagine (D148N) partially restored cell surface expression, although no specific ligand-binding or inositol phosphate signaling was detected. In contrast, conservative (D148E) substitution was expressed moderately higher, bound ligands, and signaled similarly to a hemagglutinin epitope-tagged wild-type receptor. However, D148E showed a greater tendency to be internalized once it was delivered to the membrane. Individual replacements of the conserved arginine and tyrosine (R149A, Y150A) led to decreased signal transduction without affecting surface expression, agonist affinity, or internalization or increasing basal signaling activity. Incorporation of aspartate (R149D) or reversal of charges (D148R/R149D) were nonfunctional, localized intracellularly, and indicated the absence of an ionic interaction between Asp-148 and Arg-149. It is noteworthy that an important role of arginine was identified for regulating agonist-mediated internalization when a histidine (R149H) was present. This mutant was expressed on the cell surface but was rapidly internalized after agonist treatment. This study highlights the importance of specific charged residues within this motif that provide important determinants for cell surface delivery, internalization and for normal V(1a)R function.

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 (1.35) is critical for arginine vasopressin binding. The mutant [E54A]V1aR exhibited decreased arginine vasopressin affinity (1700-fold) and disrupted signaling, but antagonist binding was unaffected. Mutation of Glu54 had an almost identical pharmacological effect as mutation of Arg46, raising the possibility that agonist binding required a mutual interaction between Glu54 and Arg46. The role of these two charged residues was investigated by 1) substituting Glu54; 2) inserting additional Glu/Arg in transmembrane helix (TM) 1; 3) repositioning the Glu/Arg in TM1; and 4) characterizing the reciprocal mutant [R46E/E54R]V1aR. We conclude that 1) the positive/negative charges need to be precisely positioned in this N terminus/TM1 segment; and 2) Glu54 and Arg46 function independently, providing two discrete epitopes required for high-affinity agonist binding and signaling. This study explains why Glu and Arg, part of an -R(X3)L/V(X3)E(X3)L- motif, are conserved at these loci throughout this G protein-coupled receptor subfamily and provides molecular insight into key differences between agonist and antagonist binding requirements.

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. The mutant receptor [A318G]OTR was engineered and characterised using ligands representing many different chemical classes. Of all the ligands investigated, only the benzoxazinone-based antagonists had decreased affinity for [A318G]OTR. Molecular modelling revealed that Ala(318) provides a direct hydrophobic contact with a methoxy group of L-371,257 and L-372,662.

An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist.

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V1aR is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V1aR mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of Ki to EC50, an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V1aR to become activated. Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V1aR. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors.