Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the “weep” response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.

Selective inhibition of the RGD (Arg-Gly-Asp) integrin αvβ1 has been recently identified as an attractive therapeutic approach for the treatment of liver fibrosis given its function, target expression, and safety profile. Our identification of a non-RGD small molecule lead followed by focused, systematic changes to the core structure utilizing a crystal structure, in silico modeling, and a tractable synthetic approach resulted in the identification of a potent small molecule exhibiting a remarkable affinity for αvβ1 relative to several other integrin isoforms measured. Azabenzimidazolone 25 demonstrated antifibrotic efficacy in an in vivo rat liver fibrosis model and represents a tool compound capable of further exploring the biological consequences of selective αvβ1 inhibition.

Tuft cells are solitary chemosensory epithelial cells that can sense lumenal stimuli at mucosal barriers and secrete effector molecules to regulate the physiology and immune state of their surrounding tissue. In the small intestine, tuft cells detect parasitic worms (helminths) and microbe-derived succinate, and signal to immune cells to trigger a Type 2 immune response that leads to extensive epithelial remodeling spanning several days. Acetylcholine (ACh) from airway tuft cells has been shown to stimulate acute changes in breathing and mucocilliary clearance, but its function in the intestine is unknown. Here we show that tuft cell chemosensing in the intestine leads to release of ACh, but that this does not contribute to immune cell activation or associated tissue remodeling. Instead, tuft cell-derived ACh triggers immediate fluid secretion from neighboring epithelial cells into the intestinal lumen. This tuft cell-regulated fluid secretion is amplified during Type 2 inflammation, and helminth clearance is delayed in mice lacking tuft cell ACh. The coupling of the chemosensory function of tuft cells with fluid secretion creates an epithelium-intrinsic response unit that effects a physiological change within seconds of activation. This response mechanism is shared by tuft cells across tissues, and serves to regulate the epithelial secretion that is both a hallmark of Type 2 immunity and an essential component of homeostatic maintenance at mucosal barriers.

Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.

This publication details the successful use of FBDD (fragment-based drug discovery) principles in the invention of a novel covalent Bruton's tyrosine kinase inhibitor, which ultimately became the Takeda Pharmaceuticals clinical candidate TAK-020. Described herein are the discovery of the fragment 5-phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one, the subsequent optimization of this hit molecule to the candidate, and synthesis and performance in pharmacodynamic and efficacy models along with direct biophysical comparison of TAK-020 with other clinical-level assets and the marketed drug Ibrutinib.

The Ca 2+ -activated TRPM5 channel plays an essential role in the perception of sweet, bitter, and umami stimuli in type II taste cells and in insulin secretion by pancreatic beta cells 1–3 . Interestingly, the voltage dependence of TRPM5 in taste bud cells depends on the intracellular Ca 2+ concentration 4 , yet the mechanism remains elusive. Here we report cryo-electron microscopy structures of the zebrafish TRPM5 in an apo closed state, a Ca 2+ -bound open state, and an antagonist-bound inhibited state, at resolutions up to 2.3 Å. We defined two novel ligand binding sites: a Ca 2+ binding site (Ca ICD ) in the intracellular domain (ICD), and an antagonist binding site in the transmembrane domain (TMD) for a drug (NDNA) that regulates insulin and GLP-1 release 5 . The Ca ICD site is unique to TRPM5 and has two roles: shifting the voltage dependence toward negative membrane potential, and promoting Ca 2+ binding to the Ca TMD site that is conserved throughout Ca 2+ -sensitive TRPM channels 6 . Replacing glutamate 337 in the Ca ICD site with an alanine not only abolished Ca 2+ binding to Ca ICD but also reduced Ca 2+ binding affinity to Ca TMD , suggesting a cooperativity between the two sites. We have defined mechanisms underlying channel activation and inhibition. Conformational changes initialized from both Ca 2+ sites, 70 Å apart, are propagated to the ICD–TMD interface and cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the TMD in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents to treat metabolic disorders.

Two different signaling pathways lead to the activation of the transcription factor NF-κB, initiating distinct biological responses: The canonical NF-κB pathway activation has been implicated in host immunity and inflammatory responses, whereas the noncanonical pathway activation has been involved in lymphoid organ development and B-cell maturation, as well as in the development of chronic inflammatory diseases and some hematologic cancers. The NF-κB-inducing kinase (NIK) is a cytoplasmic Ser/Thr kinase and is a key regulator of the noncanonical pathway. NIK activation results in the processing of the p100 subunit to p52, leading to the formation of the RelB/p52 complex and noncanonical pathway activation. Because of its role in the development of lymphoid malignancies, this kinase has always been considered as an attractive target for the treatment of certain types of cancers and immune diseases. We at Takeda have pursued a drug discovery program to identify small-molecule inhibitors against NIK. This report provides an overview of the data generated from our screening campaign using a small fragment library. Most importantly, we also provide a kinetic analysis of published compounds and chemical series developed at Takeda that are associated with a slow tight-binding mechanism and excellent cellular potency.

The Ca2+-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca2+ regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca2+ site (CaICD) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The CaICD site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca2+ binding to the CaTMD site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca2+ sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1–S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents. Cryo-EM structures of zebrafish TRPM5 reveal closed and Ca2+-bound open states, a unique Ca2+ binding site that modulates voltage sensitivity and the mechanism of antagonist action.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.

Apoptosis Signal-Regulating Kinase-1 (ASK1) is a known member of the Mitogen-Activated Protein Kinase Kinase Kinase (MAP3K) family and upon stimulation will activate the p38- and JNK-pathways leading to cardiac apoptosis, fibrosis, and hypertrophy. Using Structure-Based Drug Design (SBDD) in parallel with deconstruction of a published compound, a novel series of ASK1 inhibitors was optimized, which incorporated a saturated heterocycle proximal to the hinge-binding motif. This yielded a unique chemical series with excellent selectivity across the broader kinome, and desirable drug-like properties. The lead compound (10) is highly soluble and permeable, and exhibits a cellular EC50 = 24 nM and Kd < 1 nM. Of the 350 kinases tested, 10 has an IC50 ≤ 500 nM for only eight of them. This paper will describe the design hypotheses behind this series, key data points during the optimization phase, as well as a possible structural rationale for the kinome selectivity. Based on crystallographic data, the presence of an aliphatic cycle adjacent to the hinge-binder in the active site of the protein kinase showed up in <1% of the >5000 structures in the Protein Data Bank, potentially conferring the selectivity seen in this series.

[This corrects the article DOI: 10.1021/acsmedchemlett.6b00481.].

Guided by co-crystal structural information obtained from a different series we were exploring, a scaffold morphing and SBDD approach led to the discovery of the 1,4-disubstituted indazole series as a novel class of GKAs that potently activate GK in enzyme and cell assays. anti-diabetic OGTT efficacy was demonstrated with 29 in a rodent models of type 2 diabetes.

Using SBDD, a series of 4-amino-7-azaindoles were discovered as a novel class of Alk5 inhibitors that are potent in both Alk5 enzymatic and cellular assays. Subsequently a ring cyclization strategy was utilized to improve ADME properties leading to the discovery of a series of 1H-imidazo[4,5-c]pyridin-2(3H)-one drug like Alk5 inhibitors.

L'invention concerne des composes de formule 1 qui sont utiles comme inhibiteurs de TBK1, des compositions pharmaceutiques les contenant, des methodes de traitement d'etats associes au TBK1, et des procedes de preparation de ces composes et d'intermediaires de ceux-ci.

The discovery and optimization of a series of 4-aminocinnoline-3-carboxamide inhibitors of Bruton's tyrosine kinase are reported. A fragment-based screening approach incorporating X-ray co-crystallography was used to identify a cinnoline fragment and characterize its binding mode in the ATP binding site of Btk. Optimization of the fragment hit resulted in the identification of a lead compound which reduced paw swelling in a dose- and exposure-dependent fashion in a rat model of collagen-induced arthritis.

A compound of Formula 1, Formula ** ** a tautomer thereof or a pharmaceutically acceptable salt the compound or tautomer, wherein: R1 is selected from hydrogen, halo, -CN, C1-4 alkyl, C1-4 haloalkyl and -OR14; each of R2 and R3 is independently selected from hydrogen, halo, -CN, R6, and R7, or R2 and R3 together with the carbon atoms to which they are attached form a benzene ring or a pyridine ring in wherein the benzene ring is optionally substituted with one to four substituents independently selected from halo, -CN, R6 and R7, and the pyridine ring is optionally substituted with one to three substituents independently selected from halo, -CN, R6 and R7; R4 has the formula wherein indicates a point of attachment; L is selected from -O-, -CH2O- and -N (R4e) -; R4a is selected from -CH2R5 and ethenyl optionally substituted with one to three substituents independently selected from halo, cyano and R7; and (a) R4c is hydrogen, R 4e is selected from hydrogen and C1-4alkyl when L is -N (R4e) -, R4b and R4d and, together with a nitrogen atom and the carbon atoms to which R4b, R4c and R4d are respectively attached, form a pyrrolidine ring or a piperidine ring, each ring optionally substituted with one to six substituents independently selected from halo, C1-4 alkyl and C1-4 haloalkyl; or (b) R4b is selected from hydrogen and C1-4alkyl, R4d is hydrogen, L is -N (R4e) -, and R4c and R4e together with the carbon atoms and a nitrogen atom to which R4c, R4d and R4e are respectively attached, form a pyrrolidine ring or a piperidine ring, each ring optionally substituted with one to six substituents independently selected from halo, C1-4 alkyl and C1-4 haloalkyl; or (c) R4d is hydrogen, R 4e is selected from hydrogen and C1-4alkyl when L is -N (R4e) -, and 4b and R 4c together with the nitrogen and carbon atoms to which 4b and R 4c are respectively form a pyrrolidine ring or a piperidine ring, each ring optionally substituted with one to six substituents independently selected from halo, C1-4 alkyl and C1-4 haloalkyl; R5 is selected from hydrogen, halo and C1-4alkyl; each R6 is independently selected from -OR8, -N (R8) R9, -NR 8 C (O) R9, -NHC (O) NR8R9, - NR 8 C (O) NHR 9, -C (O) R8, -C (O) OR8 , -C (O) N (R8) R9, -C (O) N (R8) OR9, -C (O) N (R8) S (O) 2R7, -N (R8) S (O) 2R7, - SR8, -S (O) R7, -S (O) 2R7 and -S (O) 2N (R8) R9; each R7 is independently selected from (a) C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, each optionally substituted with one to five substituents independently selected from halo, oxo, -CN and R10; and (b) C3-10- cycloalkyl (CH2) m-, aryl C6-14- (CH2) m-, C 2-6 heterocyclyl (CH2) m- and C1-9- heteroaryl (CH2) m-, each optionally substituted with one to five substituents independently selected from halo, oxo, -CN, R10 and C1-6 alkyl optionally substituted with one to five substituents independently selected from halo, oxo, -CN and R10; each R8 and R9 is independently selected from (a) hydrogen; (B) C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, each optionally substituted with one to five substituents independently selected from halo, oxo, -CN and R10; and (c) C3-10- cycloalkyl (CH2) m-, aryl C6-14- (CH2) m-, C 2-6 heterocyclyl (CH2) m- and C1-9- heteroaryl (CH2) m-, each optionally substituted with one to five substituents independently selected from halo, oxo, -CN, R10 and C1-6 alkyl optionally substituted with one to five substituents independently selected from halo, oxo, -CN and R10; each R10 is independently selected from -OR11, -N (R11) R12, -N (R11) C (O) R12, -NHC (O) NR11R12, - NR 11 C (O) NHR12, -C (O) R11, -C (O) OR11, -C (O) N (R11) R12, -C (O) N (R11) OR12, -C (O) N (R11) S (O) 2R13, - NR11S (O) 2R13, - SR11, -S (O) R13, -S (O) 2R13 and -S (O) 2N (R11) R12; each R11 and R12 is independently selected from (a) hydrogen; and (b) C1-6alkyl and C3-10- cycloalkyl (CH2) m-, each optionally substituted with one to five substituents independently selected from halo, oxo, -CN, -OH and -NH2; each R13 is independently selected from C1-6 cycloalkyl and C3-10- (CH2) m- alkyl, each optionally substituted with one to five substituents independently selected from halo, oxo, - CN, -OH and -NH2; each R14 is independently selected from hydrogen, C1-4 alkyl and C1-4 haloalkyl; and each m is independently selected from 0, 1, 2, 3 and 4; wherein each heteroaryl and heterocyclyl R7, R8 and R9 independently has one to four heteroatoms, each heteroatom independently selected from N, O and S.