ABSTRACT
The discovery of a series of substituted diarylether compounds as retinoic acid related orphan receptor γt (RORγt) agonists is described. Compound 1 was identified from deck mining as a RORγt agonist. Hit-to-lead optimization led to the identification of lead compound 5, which possesses improved potency (10x). Extensive SAR exploration led to the identification of a potent and selective compound 22, that demonstrated an improved pharmacokinetic profile and a dose-dependent pharmacodynamic response. However, when dosed in a MC38 syngeneic tumor model, no evidence of efficacy was observed. ©2020 Elsevier Science Ltd. All rights reserved.
Subject(s)
Ethers/pharmacology , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Tretinoin/pharmacology , Animals , Crystallography, X-Ray , Dose-Response Relationship, Drug , Ethers/chemical synthesis , Ethers/chemistry , Humans , Mice , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Th17 Cells , Tretinoin/chemical synthesis , Tretinoin/chemistryABSTRACT
There was a time not that long ago when scientific communication happened only in print, with research papers, letters to the editor, and all other exchanges just appearing in issues mailed weeks or months after the papers were formally accepted. Learning about this research required a personal subscription to specific journals, a well-funded library, or attendance at conferences to hear the latest results and read newly released journal issues. Even as the web came into existence, the idea of using this new platform to make a scientific journal available around the country or even the world was simply not considered. That is, until the JBC-then under the leadership of Dr. Herb Tabor-decided they must find a way. Working with professors at Stanford and the newly formed Stanford service group, Highwire Press, "JBC Online" was born in 1995. In this article, as a tribute to Dr. Tabor's 100th birthday, I share my perspective and those of others involved in this transition as to what is so remarkable about the JBC going online. Specifically, I describe how Dr. Tabor's governance and leadership of the JBC, and within the American Society for Biochemistry and Molecular Biology, brought together many threads in the research, society, publishing, and technological context of the time to make sure that the JBC not only made this remarkable leap, but made it first and farthest, and paved a path for many other, and now virtually all, journals to follow.
Subject(s)
Access to Information , Biomedical Research , Online Systems , Periodicals as Topic/standards , Printing , Publishing/standards , HumansABSTRACT
In an effort to discover oral inverse agonists of RORγt to treat inflammatory diseases, a new 2,6-difluorobenzyl ether series of cyclopentyl sulfones were found to be surprisingly more potent than the corresponding alcohol derivatives. When combined with a more optimized phenyl ((R)-3-phenylpyrrolidin-3-yl)sulfone template, the 2,6-difluorobenzyl ethers yielded a set of very potent RORγt inverse agonists (e.g., compound 26, RORγt Gal4 EC50 11 nM) that are highly selective against PXR, LXRα and LXRß. After optimizing for stability in human and mouse liver microsomes, compounds 29 and 38 were evaluated in vivo and found to have good oral bioavailability (56% and 101%, respectively) in mice. X-ray co-crystal structure of compound 27 in RORγt revealed that the bulky benzyl ether group causes helix 11 of the protein to partially uncoil to create a new, enlarged binding site, which nicely accommodates the benzyl ether moiety, leading to net potency gain.
Subject(s)
Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , Pyrrolidines/pharmacology , Sulfones/pharmacology , Animals , Crystallography, X-Ray , Drug Discovery , Drug Inverse Agonism , Drug Stability , Hep G2 Cells , Humans , Mice , Microsomes, Liver/metabolism , Molecular Structure , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Pyrrolidines/chemical synthesis , Pyrrolidines/metabolism , Structure-Activity Relationship , Sulfones/chemical synthesis , Sulfones/metabolismABSTRACT
Substituted benzyloxy aryl compound 2 was identified as an RORγt agonist. Structure based drug design efforts resulted in a potent and selective tricyclic compound 19 which, when administered orally in an MC38 mouse tumor model, demonstrated a desired pharmacokinetic profile as well as a dose-dependent pharmacodynamic response. However, no perceptible efficacy was observed in this tumor model at the doses investigated.
Subject(s)
Benzyl Compounds/pharmacology , Heterocyclic Compounds/pharmacology , Receptors, Retinoic Acid/agonists , Animals , Benzyl Compounds/chemistry , Dose-Response Relationship, Drug , Female , Heterocyclic Compounds/chemistry , Mice , Mice, Inbred C57BL , Molecular Structure , Structure-Activity Relationship , Retinoic Acid Receptor gammaABSTRACT
A novel series of cis-3,4-diphenylpyrrolidines were designed as RORγt inverse agonists based on the binding conformation of previously reported bicyclic sulfonamide 1. Preliminary synthesis and structure-activity relationship (SAR) study established (3S,4S)-3-methyl-3-(4-fluorophenyl)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenyl)pyrrolidine as the most effective scaffold. Subsequent SAR optimization led to identification of a piperidinyl carboxamide 31, which was potent against RORγt (EC50 of 61 nM in an inverse agonist assay), selective relative to RORα, RORß, LXRα and LXRß, and stable in human and mouse liver microsomes. Furthermore, compound 31 exhibited considerably lower PXR Ymax (46%) and emerged as a promising lead. The binding mode of the diphenylpyrrolidine series was established with an X-ray co-crystal structure of 10A/RORγt.
Subject(s)
Drug Design , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Pyrrolidines/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Drug Inverse Agonism , Humans , Mice , Microsomes, Liver/metabolism , Molecular Dynamics Simulation , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Pregnane X Receptor/agonists , Pregnane X Receptor/metabolism , Pyrrolidines/chemical synthesis , Pyrrolidines/metabolism , Structure-Activity RelationshipABSTRACT
Structure-activity relationship optimization on a series of phenylpyrazole amides led to the identification of a dual ROCK1 and ROCK2 inhibitor (25) which demonstrated good potency, kinome selectivity and favorable pharmacokinetic profiles. Compound 25 was selected as a tool molecule for in vivo studies including evaluating hemodynamic effects in telemeterized mice, from which moderate decreases in blood pressure were observed.
Subject(s)
Amides/pharmacology , Drug Discovery , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Amides/chemical synthesis , Amides/chemistry , Animals , Blood Pressure/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Mice , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Structure-Activity Relationship , rho-Associated Kinases/metabolismABSTRACT
In order to rapidly develop C6 and C8 SAR of our reported tricyclic sulfone series of RORγt inverse agonists, a late-stage bromination was employed. Although not regioselective, the bromination protocol allowed us to explore new substitution patterns/vectors that otherwise would have to be incorporated at the very beginning of the synthesis. Based on the SAR obtained from this exercise, compound 15 bearing a C8 fluorine was developed as a very potent and selective RORγt inverse agonist. This analog's in vitro profile, pharmacokinetic (PK) data and efficacy in an IL-23 induced mouse acanthosis model will be discussed.
Subject(s)
Heterocyclic Compounds, 3-Ring/therapeutic use , Melanosis/drug therapy , Nuclear Receptor Subfamily 1, Group F, Member 3/antagonists & inhibitors , Sulfones/therapeutic use , Animals , Crystallography, X-Ray , Drug Inverse Agonism , Female , Heterocyclic Compounds, 3-Ring/chemical synthesis , Heterocyclic Compounds, 3-Ring/pharmacokinetics , Interleukin-18 , Male , Melanosis/chemically induced , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Structure , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Protein Binding , Structure-Activity Relationship , Sulfones/chemical synthesis , Sulfones/pharmacokineticsABSTRACT
An X-ray crystal structure of one of our previously discovered RORγt inverse agonists bound to the RORγt ligand binding domain revealed that the cyclohexane carboxylic acid group of compound 2 plays a significant role in RORγt binding, forming four hydrogen bonding and ionic interactions with RORγt. SAR studies centered around the cyclohexane carboxylic acid group led to identification of several structurally diverse and more potent compounds, including new carboxylic acid analogues 7 and 20, and cyclic sulfone analogues 34 and 37. Notably, compounds 7 and 20 were found to maintain the desirable pharmacokinetic profile of 2.
Subject(s)
Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Pyrrolidines/pharmacology , Sulfones/pharmacology , Administration, Oral , Animals , Biological Availability , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Inverse Agonism , Humans , Mice , Models, Molecular , Molecular Structure , Pyrrolidines/administration & dosage , Pyrrolidines/chemistry , Structure-Activity Relationship , Sulfones/administration & dosage , Sulfones/chemistryABSTRACT
We disclose the optimization of a high throughput screening hit to yield benzothiazine and tetrahydroquinoline sulfonamides as potent RORγt inverse agonists. However, a majority of these compounds showed potent activity against pregnane X receptor (PXR) and modest activity against liver X receptor α (LXRα). Structure-based drug design (SBDD) led to the identification of benzothiazine and tetrahydroquinoline sulfonamide analogs which completely dialed out LXRα activity and were less potent at PXR. Pharmacodynamic (PD) data for compound 35 in an IL-23 induced IL-17 mouse model is discussed along with the implications of a high Ymax in the PXR assay for long term preclinical pharmacokinetic (PK) studies.
Subject(s)
Bridged Bicyclo Compounds/pharmacology , Drug Design , Propanols/pharmacology , Receptors, Retinoic Acid/agonists , Receptors, Steroid/agonists , Sulfonamides/pharmacology , Animals , Bridged Bicyclo Compounds/chemical synthesis , Bridged Bicyclo Compounds/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Liver X Receptors/agonists , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Pregnane X Receptor , Propanols/chemical synthesis , Propanols/chemistry , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Retinoic Acid Receptor gammaABSTRACT
The identification of small molecule inhibitors of IRAK4 for the treatment of autoimmune diseases has been an area of intense research. We discovered novel 4,6-diaminonicotinamides which potently inhibit IRAK4. Optimization efforts were aided by X-ray crystal structures of inhibitors bound to IRAK4. Structure activity relationship (SAR) studies led to the identification of compound 29 which exhibited sub-micromolar potency in a LTA stimulated cellular assay.
Subject(s)
Drug Design , Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Niacinamide/chemistry , Protein Kinase Inhibitors/chemistry , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Interleukin-1 Receptor-Associated Kinases/metabolism , Janus Kinase 3/chemistry , Janus Kinase 3/metabolism , Molecular Conformation , Molecular Dynamics Simulation , Niacinamide/metabolism , Protein Kinase Inhibitors/metabolism , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.
Subject(s)
Janus Kinase 1/antagonists & inhibitors , Janus Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridazines/chemistry , Pyrroles/chemistry , Animals , Binding Sites , Catalytic Domain , Cell Line , Crystallography, X-Ray , Disease Models, Animal , Drug Evaluation, Preclinical , Half-Life , Humans , Inflammation/prevention & control , Inhibitory Concentration 50 , Janus Kinase 1/metabolism , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Janus Kinase 3/metabolism , Mice , Mice, Inbred BALB C , Molecular Conformation , Molecular Dynamics Simulation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Pyridazines/chemical synthesis , Pyridazines/pharmacokinetics , Pyrroles/chemical synthesis , Pyrroles/pharmacokinetics , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , TYK2 Kinase/antagonists & inhibitors , TYK2 Kinase/metabolismABSTRACT
As demonstrated in preclinical animal models, the disruption of PI3Kδ expression or its activity leads to a decrease in inflammatory and immune responses. Therefore, inhibition of PI3Kδ may provide an alternative treatment for autoimmune diseases, such as RA, SLE, and respiratory ailments. Herein, we disclose the identification of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent, selective and orally bioavailable PI3Kδ inhibitors. The lead compound demonstrated efficacy in an in vivo mouse KLH model.
Subject(s)
Amines/chemistry , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Amines/metabolism , Amines/therapeutic use , Animals , Autoimmune Diseases/drug therapy , Binding Sites , Class I Phosphatidylinositol 3-Kinases , Crystallography, X-Ray , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Mice , Microsomes, Liver/metabolism , Molecular Docking Simulation , Phosphatidylinositol 3-Kinases/metabolism , Piperazine , Piperazines/chemistry , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/therapeutic use , Structure-Activity Relationship , Triazines/chemistryABSTRACT
A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3 active site cysteine residue. The design was based on crystal structure information and a comparative study of several electrophilic warheads.
Subject(s)
Janus Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Binding Sites , Catalytic Domain , Cell Line , Cell Survival/drug effects , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 1/metabolism , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Janus Kinase 3/metabolism , Molecular Docking Simulation , Protein Kinase Inhibitors/metabolism , Structure-Activity RelationshipABSTRACT
Aberrant Class I PI3K signaling is a key factor contributing to many immunological disorders and cancers. We have identified 4-amino pyrrolotriazine as a novel chemotype that selectively inhibits PI3Kδ signaling despite not binding to the specificity pocket of PI3Kδ isoform. Structure activity relationship (SAR) led to the identification of compound 30 that demonstrated efficacy in mouse Keyhole Limpet Hemocyanin (KLH) and collagen induced arthritis (CIA) models.
Subject(s)
Amines/chemistry , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Triazines/chemistry , Amines/metabolism , Amines/therapeutic use , Animals , Arthritis/drug therapy , Arthritis/metabolism , Arthritis/pathology , Binding Sites , Disease Models, Animal , Drug Evaluation, Preclinical , Inhibitory Concentration 50 , Mice , Molecular Docking Simulation , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
The discovery, synthesis, and characterization of 9H-carbazole-1-carboxamides as potent and selective ATP-competitive inhibitors of Janus kinase 2 (JAK2) are discussed. Optimization for JAK family selectivity led to compounds 14 and 21, with greater than 45-fold selectivity for JAK2 over all other members of the JAK kinase family.
Subject(s)
Amides/chemistry , Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Amides/metabolism , Amides/pharmacology , Binding Sites , Carbazoles/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Humans , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 1/metabolism , Janus Kinase 2/metabolism , Janus Kinase 3/antagonists & inhibitors , Janus Kinase 3/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g-4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3-JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3-JAK1 pathway versus JAK2, and active in a human whole blood assay.
Subject(s)
Drug Discovery , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyridazines/chemistry , Pyrroles/chemistry , Administration, Oral , Animals , Enzyme-Linked Immunosorbent Assay , Humans , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Protein Conformation/drug effects , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity Relationship , Tissue DistributionABSTRACT
A novel series of p38 MAP kinase inhibitors with high selectivity for the p38α isoform over the other family members including the highly homologous p38ß isoform has been identified. X-ray co-crystallographic studies have revealed an unprecedented kinase binding mode in p38α for representative analogs, 5c and 9d, in which a Leu108/Met109 peptide flip occurs within the p38α hinge region. Based on these findings, a general strategy for the rational design of additional promising p38α isoform selective inhibitors by targeting this novel binding mode is proposed.
Subject(s)
Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Binding Sites , Crystallography, X-Ray , Humans , Hydrogen Bonding , Mitogen-Activated Protein Kinase 14/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure confirmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.
Subject(s)
Antineoplastic Agents/chemical synthesis , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , Sulfonamides/chemical synthesis , bcl-2-Associated X Protein/antagonists & inhibitors , bcl-X Protein/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Cytochromes c/metabolism , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Models, Molecular , Protein Binding , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Sulfonamides/pharmacology , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , bcl-X Protein/chemistry , bcl-X Protein/metabolismABSTRACT
CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.
Subject(s)
Indoles/antagonists & inhibitors , Indoles/chemistry , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/chemistry , Pyrazoles/antagonists & inhibitors , Pyrazoles/chemistry , Amino Acid Sequence , Catalytic Domain/drug effects , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Indoles/metabolism , Molecular Sequence Data , Protein Binding/drug effects , Protein-Arginine N-Methyltransferases/metabolism , Pyrazoles/metabolismABSTRACT
The X-ray crystal structure of a human cardiac muscle troponin C/troponin I chimera has been determined in two different crystal forms and shows a conformation of the complex that differs from that previously observed by NMR. The chimera consists of the N-terminal domain of troponin C (cTnC; residues 1-80) fused to the switch region of troponin I (cTnI; residues 138-162). In both crystal forms, the cTnI residues form a six-turn α-helix that lays across the hydrophobic groove of an adjacent cTnC molecule in the crystal structure. In contrast to previous models, the cTnI helix runs in a parallel direction relative to the cTnC groove and completely blocks the calcium desensitizer binding site of the cTnC-cTnI interface.