Identification of imidazo[1,2-b]pyridazine TYK2 pseudokinase ligands as potent and selective allosteric inhibitors of TYK2 signalling.

As a member of the Janus (JAK) family of non-receptor tyrosine kinases, TYK2 mediates the signaling of pro-inflammatory cytokines including IL-12, IL-23 and type 1 interferon (IFN), and therefore represents an attractive potential target for treating the various immuno-inflammatory diseases in which these cytokines have been shown to play a role. Following up on our previous report that ligands to the pseudokinase domain (JH2) of TYK2 suppress cytokine-mediated receptor activation of the catalytic (JH1) domain, the imidazo[1,2-b]pyridazine (IZP) 7 was identified as a promising hit compound. Through iterative modification of each of the substituents of the IZP scaffold, the cellular potency was improved while maintaining selectivity over the JH1 domain. These studies led to the discovery of the JH2-selective TYK2 inhibitor 29, which provided encouraging systemic exposures after oral dosing in mice. Phosphodiesterase 4 (PDE4) was identified as an off-target and potential liability of the IZP ligands, and selectivity for TYK2 JH2 over this enzyme was obtained by elaborating along selectivity vectors determined from analyses of X-ray co-crystal structures of representative ligands of the IZP class bound to both proteins.

Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

Quinazolines as cyclin dependent kinase inhibitors.

Quinazolines have been identified as inhibitors of CDK4/D1 and CDK2/E. Aspects of the SAR were investigated using solution-phase, parallel synthesis. An X-ray crystal structure was obtained of quinazoline 51 bound in CDK2 and key interactions within the ATP binding pocket are defined.

Structure determination of coxsackievirus B3 to 3.5 A resolution.

The crystal structure of coxsackievirus B3 (CVB3) has been determined to 3.5 A resolution. The icosahedral CVB3 particles crystallize in the monoclinic space group, P2(1), (a = 574.6, b = 302.1, c = 521.6 A, beta = 107.7 degrees ) with two virions in the asymmetric unit giving 120-fold non-crystallographic redundancy. The crystals diffracted to 2.7 A resolution and the X-ray data set was 55% complete to 3.0,4, resolution. Systematically weak reflections and the self-rotation function established pseudo R32 symmetry with each particle sitting on a 32 special position. This constrained the orientation and position of each particle in the monoclinic cell to near face-centered positions and allowed for a total of six possible monoclinic space-group settings. Correct interpretation of the high-resolution (3.0-3.2 A) self-rotation function was instrumental in determining the deviations from R32 orientations of the virus particles in the unit cell. Accurate particle orientations permitted the correct assignment of the crystal space-group setting amongst the six ambiguous possibilities and for the correct determination of particle positions. Real-space electron-density averaging and phase refinement, using human rhinovius 14 (HRV14) as an initial phasing model, have been carried out to 3.5 A resolution. The initial structural model has been built and refined to 3.5 A resolution using X-PLOR.

Phase refinement and extension by means of non-crystallographic symmetry averaging using parallel computers.

Electron-density averaging, fast Fourier synthesis and fast Fourier analysis programs have been adapted for parallel-computing systems. These have been linked to perform iterative phase improvement and extension utilizing non-crystallographic symmetry and solvent flattening. Various strategies for parallel algorithms have been tested on a variety of computers as a function of the number of computer nodes. Some experimental timing results are discussed.

The structure of coxsackievirus B3 at 3.5 A resolution.

BACKGROUND: Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs. RESULTS: The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 A. The beta-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 beta-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes. CONCLUSIONS: The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.

Human rhinovirus 14 complexed with fragments of active antiviral compounds.

Crystallographic studies of human rhinovirus 14 (HRV14) crystals soaked with fragments of antiviral WIN compounds, at high concentrations (82-200 micrograms/ml), show the compounds bind into the hydrophobic beta-barrel (WIN pocket) of VP1. Two of these short compounds (5-[3,5-dimethyl-4-hydroxyphenyl]-2-methyltetrazole and phenol oxazoline) cause conformational changes in the virus similar to the active, longer WIN compounds. In addition, thermostabilization studies suggest these short WIN compounds provide some stability to the HRV14 capsid. We conclude that the short compounds appear to mimic the cellular cofactors observed in the hydrophobic pocket of VP1 for some picornaviruses. Both cofactors and short WIN compounds bind into the pocket, cause conformational changes in VP1, and provide a small degree of virion stabilization but are unlikely to inhibit attachment. Three specific binding sites for dimethyl sulfoxide (DMSO), used as solvent, were also identified. One of the DMSO molecules binds into the drug binding pocket near the pocket opening, while the other two bind in the canyon near the VP1 protomer-protomer interface.