Structure-activity relationship study of central pyridine-derived TYK2 JH2 inhibitors: Optimization of the PK profile through C4' and C6 variations.

Efforts directed at improving potency and preparing structurally different TYK2 JH2 inhibitors from the first generation of compounds such as 1a led to the SAR study of new central pyridyl based analogs 2-4. The current SAR study resulted in the identification of 4h as a potent and selective TYK2 JH2 inhibitor with distinct structural differences from 1a. In this manuscript, the in vitro and in vivo profiles of 4h are described. The hWB IC50 of 4h was shown as 41 nM with 94% bioavailability in the mouse PK study.

Discovery of BMS-986202: A Clinical Tyk2 Inhibitor that Binds to Tyk2 JH2.

A search for structurally diversified Tyk2 JH2 ligands from 6 (BMS-986165), a pyridazine carboxamide-derived Tyk2 JH2 ligand as a clinical Tyk2 inhibitor currently in late development for the treatment of psoriasis, began with a survey of six-membered heteroaryl groups in place of the N-methyl triazolyl moiety in 6. The X-ray co-crystal structure of an early lead (12) revealed a potential new binding pocket. Exploration of the new pocket resulted in two frontrunners for a clinical candidate. The potential hydrogen bonding interaction with Thr599 in the pocket was achieved with a tertiary amide moiety, confirmed by the X-ray co-crystal structure of 29. When the diversity search was extended to nicotinamides, a single fluorine atom addition was found to significantly enhance the permeability, which directly led to the discovery of 7 (BMS-986202) as a clinical Tyk2 inhibitor that binds to Tyk2 JH2. The preclinical studies of 7, including efficacy studies in mouse models of IL-23-driven acanthosis, anti-CD40-induced colitis, and spontaneous lupus, will also be presented.

A Novel Peptidylarginine Deiminase 4 (PAD4) Inhibitor BMS-P5 Blocks Formation of Neutrophil Extracellular Traps and Delays Progression of Multiple Myeloma.

Multiple myeloma is a plasma cell malignancy, which grows in the bone marrow (BM). The major population of cells in the BM is represented by neutrophils and they can form neutrophil extracellular traps (NET). Here, we investigated whether multiple myeloma cells induce NET formation and whether targeting this process would delay multiple myeloma progression. We demonstrated that murine and human multiple myeloma cells stimulate citrullination of histone H3 and NET formation by neutrophils and that this process is abrogated by pharmacological targeting of peptidylarginine deiminase 4 (PAD4) with a novel-specific small molecule inhibitor BMS-P5. Administration of BMS-P5 to multiple myeloma-bearing mice delays appearance of symptoms and disease progression. Taken together, our data demonstrate that targeting PAD4 may be beneficial for treatment of multiple myeloma.

Identification of N-Methyl Nicotinamide and N-Methyl Pyridazine-3-Carboxamide Pseudokinase Domain Ligands as Highly Selective Allosteric Inhibitors of Tyrosine Kinase 2 (TYK2).

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165.

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain.

TYK2 is a nonreceptor tyrosine kinase involved in adaptive and innate immune responses. A deactivating coding variant has previously been shown to prevent receptor-stimulated activation of this kinase and provides high protection from several common autoimmune diseases but without immunodeficiency. An agent that recapitulates the phenotype of this deactivating coding variant may therefore represent an important advancement in the treatment of autoimmunity. BMS-986165 is a potent oral agent that similarly blocks receptor-stimulated activation of TYK2 allosterically and with high selectivity and potency afforded through optimized binding to a regulatory domain of the protein. Signaling and functional responses in human TH17, TH1, B cells, and myeloid cells integral to autoimmunity were blocked by BMS-986165, both in vitro and in vivo in a phase 1 clinical trial. BMS-986165 demonstrated robust efficacy, consistent with blockade of multiple autoimmune pathways, in murine models of lupus nephritis and inflammatory bowel disease, supporting its therapeutic potential for multiple immune-mediated diseases.

Identification of Imidazo[1,2-b]pyridazine Derivatives as Potent, Selective, and Orally Active Tyk2 JH2 Inhibitors.

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

Purine derivatives as potent Bruton's tyrosine kinase (BTK) inhibitors for autoimmune diseases.

Investigation of various heterocyclic core isosteres of imidazopyrazines 1 & 2 yielded purine derivatives 3 & 8 as potent and selective BTK inhibitors. Subsequent SAR studies of the purine series led to the discovery of 20 as a leading compound. Compound 20 is very selective when screened against a panel of 400 kinases and is a potent inhibitor in cellular assays of human B cell function including B-Cell proliferation and CD86 cell surface expression and exhibited in vivo efficacy in a mouse PCA model. Its X-ray co-crystal structure with BTK shows that the high selectivity is gained from filling a BTK specific lipophilic pocket. However, physical and ADME properties leading to low oral exposure hindered further development.

Periodic, partial inhibition of IkappaB Kinase beta-mediated signaling yields therapeutic benefit in preclinical models of rheumatoid arthritis.

We have previously shown that inhibitors of IkappaB kinase beta (IKKbeta), including 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline (BMS-345541), are efficacious against experimental arthritis in rodents. In our efforts to identify an analog as a clinical candidate for the treatment of autoimmune and inflammatory disorders, we have discovered the potent and highly selective IKKbeta inhibitor 2-methoxy-N-((6-(1-methyl-4-(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-7-yl)pyridin-2-yl)methyl)acetamide (BMS-066). Investigations of its pharmacology in rodent models of experimental arthritis showed that BMS-066 at doses of 5 and 10 mg/kg once daily was effective at protecting rats against adjuvant-induced arthritis, despite showing only weak inhibition at 10 mg/kg against a pharmacodymanic model of tumor necrosis factor alpha production in rats challenged with lipopolysaccharide. The duration of exposure in rats indicated that just 6 to 9 h of coverage per day of the concentration necessary to inhibit IKKbeta by 50% in vivo was necessary for protection against arthritis. Similar findings were observed in the mouse collagen-induced arthritis model, with efficacy observed at a dose providing only 6 h of coverage per day of the concentration necessary to inhibit IKKbeta by 50%. This finding probably results from the cumulative effect on multiple cellular mechanisms that contribute to autoimmunity and joint destruction, because BMS-066 was shown to inhibit a broad spectrum of activities such as T cell proliferation, B cell function, cytokine and interleukin secretion from monocytes, T(H)17 cell function and regulation, and osteoclastogenesis. Thus, only partial and transient inhibition of IKKbeta is sufficient to yield dramatic benefit in vivo, and this understanding will be important in the clinical development of IKKbeta inhibitors.

IkappaB kinase inhibitors for treating autoimmune and inflammatory disorders: potential and challenges.

The nuclear transcription factor NF-kappaB has a crucial role in the pathogenesis of several human disorders, particularly those with an inflammatory component. Because the multisubunit IkappaB kinase (IKK) is vital for translating pro-inflammatory stimuli into the activation of NF-kappaB, this kinase provides an opportunity to develop novel therapeutics. In this article, we review the investigations, both genetic and pharmacological, that demonstrate the use of IKK inhibition in autoimmune and inflammatory disorders. It is still unclear what toxicities will be associated with IKK inhibitors; a discussion of the potential for mechanism-based toxicities such as teratogenicity, lymphopoietic defects and susceptibility to infection is also presented.

NEMO binding domain of IKK-2 encompasses amino acids 735-745.

NF-kappaB activation is mediated by the IKK signalsome. Though this signalsome is comprised of IKK-1, IKK-2, and NEMO/IKKgamma, it is the interaction between IKK-2 and NEMO that is critical to formation of a functional signalsome. More specifically, previous reports have indicated that this interaction involves the C-terminal LDWSWL residues of IKK-2 (called the Nemo Binding Domain (NBD)) and the N-terminus of NEMO. In an effort to characterize the IKK-2:NEMO interaction, we have investigated several NBD-containing peptides for their ability to bind NEMO and inhibit the critical IKK-2:NEMO interaction. The six residue NBD peptide, LDWSWL, showed modest binding to NEMO and little inhibition of the IKK-2:NEMO interaction, whereas peptides containing the NBD plus additional flanking amino acids (NBD-containing peptides) more effectively bound NEMO and inhibited the interaction. These longer NBD-containing peptides may be required to give the NBD an appropriate conformation for recognition by NEMO and/or to provide for additional interactions with NEMO.

BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice.

The signal-inducible phosphorylation of serines 32 and 36 of I kappa B alpha is critical in regulating the subsequent ubiquitination and proteolysis of I kappa B alpha, which then releases NF-kappa B to promote gene transcription. The multisubunit I kappa B kinase responsible for this phosphorylation contains two catalytic subunits, termed I kappa B kinase (IKK)-1 and IKK-2. BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKK-2 IC(50) = 0.3 microm, IKK-1 IC(50) = 4 microm). The compound failed to inhibit a panel of 15 other kinases and selectively inhibited the stimulated phosphorylation of I kappa B alpha in cells (IC(50) = 4 microm) while failing to affect c-Jun and STAT3 phosphorylation, as well as mitogen-activated protein kinase-activated protein kinase 2 activation in cells. Consistent with the role of IKK/NF-kappa B in the regulation of cytokine transcription, BMS-345541 inhibited lipopolysaccharide-stimulated tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and interleukin-6 in THP-1 cells with IC(50) values in the 1- to 5-microm range. Although a Dixon plot of the inhibition of IKK-2 by BMS-345541 showed a non-linear relationship indicating non-Michaelis-Menten kinetic binding, the use of multiple inhibition analyses indicated that BMS-345541 binds in a mutually exclusive manner with respect to a peptide inhibitor corresponding to amino acids 26-42 of I kappa B alpha with Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive manner with respect to ADP. The opposite results were obtained when studying the binding to IKK-1. A binding model is proposed in which BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which then affects the active sites of the subunits differently. BMS-345541 was also shown to have excellent pharmacokinetics in mice, and peroral administration showed the compound to dose-dependently inhibit the production of serum tumor necrosis factor alpha following intraperitoneal challenge with lipopolysaccharide. Thus, the compound is effective against NF-kappa B activation in mice and represents an important tool for investigating the role of IKK in disease models.

The catalytic subunits of IkappaB kinase, IKK-1 and IKK-2, contain non-equivalent active sites when expressed as homodimers.

The signal-inducible phosphorylation of serines 32 and 36 of IkappaBalpha is the key step in regulating the subsequent ubiquitination and proteolysis of IkappaBalpha which then releases NF-kappaB to promote gene transcription. The multisubunit IkappaB kinase responsible for this phosphorylation contains two catalytic subunits, termed IKK-1 and IKK-2. It has been shown that both subunits catalyze the phosphorylation of IkappaBalpha as well as an autophosphorylation at a C-terminal cluster of serines. With baculovirus/insect cell-expressed homodimeric IKK-1 or IKK-2, inhibitors such as ADP or a peptide inhibitor (corresponding to amino acid residues 26-42 of IkappaBalpha with Ser-32 and Ser-36 changed to aspartates) inhibited autophosphorylation and IkappaBalpha phosphorylation reactions with different potencies. ADP was more potent against IkappaBalpha phosphorylation as compared to autophosphorylation, while the peptide inhibitor showed the opposite effect. Pseudo-Dixon plots of the inhibition with ADP were linear while non-linear plots were obtained with the peptide inhibitor, suggesting a cooperative effect in the case of the latter. Using different concentrations of IKK-1, autophosphorylation was shown to be intramolecular. These results indicated that there were non-equivalent active sites present within the preparations of recombinant homodimers of IKK-1 and IKK-2. The peptide inhibitor showed equivalent inhibitory effects with wild-type IKK-1 and the S176E/S180E mutant. In contrast, ADP showed equipotent inhibition against the S176E/S180E mutant-catalyzed autophosphorylation and IkappaBalpha phosphorylation reactions. A model is proposed in which the phosphorylation state of the activation loop of IKK-1 or IKK-2 affects the active site conformation of the enzyme such that the two forms catalyze the autophosphorylation and IkappaBalpha phosphorylation reactions with different affinities. In addition, the two active sites within the dimer appear to act in a cooperative fashion so that binding of peptide inhibitor at one active site affects the conformation of the other active site.