No abstract

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.

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.

ABSTRACT:We explored the properties of a catenary model that includes the basolateral (B), apical (A), and cellular compartments via simulations under linear and nonlinear conditions to understand the asymmetric observations arising from transporters, enzymes, and permeability in Caco-2 cells. The efflux ratio (EfR; P app,B3A / P app,A3B ), obtained from the effective permeability from the A3B and B3A direction under linear conditions, was unity for passively permeable drugs whose transport does not involve transporters; the value was unaffected by cellular binding or metabolism, but increased with apical efflux. Metabolism was asymmetric, showing lesser metabolite accrual for the B3A than A3B direction because of inherent differences in the volumes for A and B. Moreover, the net flux (total ؊ passive permeation) due to saturable apical efflux, absorption, or metabolism showed nonconformity to simple The majority of drugs available on the market are in oral dosage forms. For the assessment of permeability and oral drug absorption, in silico models (Stenberg et al., 2001) and high throughput systems, such as the parallel artificial membrane permeability assay (Kansy et al., 1998) and cell-based systems (Hidalgo et al., 1989) exist to relate drug permeability to absorption, especially for compounds that do not undergo intestinal metabolism (Usansky and Sinko, 2005). The most popular high-throughput screening tool for drug permeability is human colon carcinoma (Caco-2) (Hidalgo et al., 1989) or transfected Madin-Darby canine kidney (Irvine et al., 1999) cells. Upon culture, Caco-2 cells differentiate and become confluent to form monolayers with tight junctions and polarized apical/mucosal (A side) and basolateral/serosal (B side) membranes that are structurally and functionally similar to those of enterocytes.ABC efflux transporters such as P-glycoprotein (Pgp) multidrug resistance-associated protein 2 (MRP2), and the breast cancer-resistant protein (BCRP) are expressed on the mucosal membrane of Caco-2 (Hunter et al., 1993;Hirohashi et al., 2000), as are the absorptive transporters, such as the proton-coupled oligopeptide transporter (PEPT1) (Guo et al., 1999) and the organic anion transporting polypeptide (OATP) (Kobayashi et al., 2003). Likewise, on the serosal membrane, basolateral efflux transporters such as MRP3 (Hirohashi et al., 2000) and organic solute transporters ␣ and ␤ (OST␣-OST␤) (Okuwaki et al., 2007) are expressed in Caco-2. In addition, multiple metabolic enzymes such as the sulfotransferases, UDPglucuronosyltransferases (H. Sun, L. Zhang, E. C. Chow, G. Lin, K. S. Pang, Z. Zuo, unpublished data), and the glutathione S-transferases (Peters and Roelofs, 1989) N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; A, apical; B, basolateral; C D,0 , initial loading concentration in donor side at t ϭ 0; P app , effective permeability; EfR, efflux ratio; MK571,vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propioni...