Andreas Kuglstatter

Selective p38α Inhibitors Clinically Evaluated for the Treatment of Chronic Inflammatory Disorders

p38R is a member of the well characterized mitogen activatedprotein (MAP) kinase familyof serine/threonineprotein kinases. p38R is widely expressed in endothelial, immune, and inflammatory cells and plays a central role in the regulation of proinflammatory cytokine production including TNF-R, IL-1β, and IL-6. Selective blockade of any one of these cytokineswith biologic agents has proven efficacious for inflammatory diseases including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. The p38 subfamily of MAP kinases includes four isoforms (p38R, p38β, p38γ, and p38δ) that are encoded by separate genes. Analysis of differential tissue expression and activation of these isoforms in synovial tissue extracted fromRApatients has suggested that the p38R isoform is overactivated within inflamed tissue andmay be a preferred target for intervention in the disease. This expectation has prompted a huge investment by thepharmaceutical industry in thedevelopment of p38R inhibitors as potential oral disease modifying antirheumatic drugs (DMARDs). This Perspective will summarize key learnings from over 15 years of industrial experience with p38R as a drug target, with a focus on the rational design of highly selective small molecule inhibitors, followed by a discussion of data for examples 1-11 that have entered into or are recruiting for phase 2 clinical studies (summarized in Table 1). To date, these results have been disappointing.We conclude that p38R inhibition alone is unlikely to be a successful strategy toward treating chronic inflammatory disorders. Others have also concluded that “the era of optimism surrounding the use of p38 MAPK inhibition for the treatment of RA is over”.

Bruton’s Tyrosine Kinase Inhibitors: Approaches to Potent and Selective Inhibition, Preclinical and Clinical Evaluation for Inflammatory Diseases and B Cell Malignancies

■ INTRODUCTION Bruton’s tyrosine kinase (BTK) is a member of the Tec tyrosine kinase family. BTK is expressed in most hematopoietic cells such as B cells, mast cells, and macrophages but not in T cells, natural killer cells, and plasma cells. BTK plays key roles in multiple cell signaling pathways including BCR and FcR signaling cascades. Mutations in the human BTK gene cause the inherited disease X-linked agammaglobulinemia (XLA), with lack of peripheral B cells and low levels of serum Ig. In XLA, the primary immune deficit is B cell specific. In fact Rituxan, a CD20 antibody, has impacted B cells on the pathogenesis of many autoimmune diseases, such as RA, SLE, and MS. This has fueled interest by multiple pharmaceutical companies in pursuing small molecule BTK inhibitors in the treatment of autoimmune diseases. Likewise, there is also interest in the development of BTK inhibitors for the treatment of hematological malignancies, as aberrant activating BTK has been implicated in the pathogenesis of B cell lymphoma. Detailed reviews and articles on BTK biology and its therapeutic potentials have been reported. Kinase selectivity is a central issue in discovering efficacious and safe small molecule inhibitors for kinase targets, especially for non-life-threatening diseases such as RA. To prevent adverse toxicological events caused by immunological responses, the pharmaceutical industry has largely focused its small molecule drug discovery efforts on agents that interact noncovalently with their target proteins. This strategy has been followed despite numerous examples of marketed drugs with target-specific covalent mode-of-action. In the case of protein kinase targets for which selectivity and efficacy pose major challenges for noncovalent inhibitors, targeted covalent inhibition has provided an attractive alternative. Both approaches, noncovalent and covalent inhibition of protein kinases, benefit tremendously from high resolution structural information from protein crystal structures. This article will review BTK structural biology with a focus on design features for selective BTK inhibitors. We first summarize the publically available structural information on the BTK kinase domain. Then we will provide a brief summary and analysis with key SAR information for the most potent inhibitors reported for the chemical classes that have been disclosed in patents and publications. When available, preclinical and clinical data for advanced compounds will be summarized.

β-Secretase (BACE1) Inhibitors with High in Vivo Efficacy Suitable for Clinical Evaluation in Alzheimer’s Disease

An extensive fluorine scan of 1,3-oxazines revealed the power of fluorine(s) to lower the pKa and thereby dramatically change the pharmacological profile of this class of BACE1 inhibitors. The CF3 substituted oxazine 89, a potent and highly brain penetrant BACE1 inhibitor, was able to reduce significantly CSF Aβ40 and 42 in rats at oral doses as low as 1 mg/kg. The effect was long lasting, showing a significant reduction of Aβ40 and 42 even after 24 h. In contrast to 89, compound 1b lacking the CF3 group was virtually inactive in vivo.

Structural insights for design of potent spleen tyrosine kinase inhibitors from crystallographic analysis of three inhibitor complexes.

Spleen tyrosine kinase is considered an attractive drug target for the treatment of allergic and antibody mediated autoimmune diseases. We have determined the co‐crystal structures of spleen tyrosine kinase complexed with three known inhibitors: YM193306, a 7‐azaindole derivative and R406. The cis‐cyclohexyldiamino moiety of YM193306 is forming four hydrophobically shielded polar interactions with the spleen tyrosine kinase protein and is therefore crucial for the high potency of this inhibitor. Its primary amino group is inducing a conformational change of the spleen tyrosine kinase DFG Asp side chain. The crystal structure of the 7‐azaindole derivative bound to spleen tyrosine kinase is the first demonstration of a 2‐substituted 7‐azaindole bound to a protein kinase. Its indole‐amide substituent is tightly packed between the N‐ and C‐terminal kinase lobes. The co‐crystal structure of the spleen tyrosine kinase–R406 complex shows two main differences to the previously reported structure of spleen tyrosine kinase soaked with R406: (i) the side chain of the highly conserved Lys is disordered and not forming a hydrogen bond to R406 and (ii) the DFG Asp side chain is pointing away from and not towards R406. The novel protein–ligand interactions and protein conformational changes revealed in these structures guide the rational design and structure‐based optimization of second‐generation spleen tyrosine kinase inhibitors.

Cutting Edge: IL-1 Receptor-Associated Kinase 4 Structures Reveal Novel Features and Multiple Conformations

IL-1R-associated kinase (IRAK)4 plays a central role in innate and adaptive immunity, and is a crucial component in IL-1/TLR signaling. We have determined the crystal structures of the apo and ligand-bound forms of human IRAK4 kinase domain. These structures reveal several features that provide opportunities for the design of selective IRAK4 inhibitors. The N-terminal lobe of the IRAK4 kinase domain is structurally distinctive due to a loop insertion after an extended N-terminal helix. The gatekeeper residue is a tyrosine, a unique feature of the IRAK family. The IRAK4 structures also provide insights into the regulation of its activity. In the apo structure, two conformations coexist, differing in the relative orientation of the two kinase lobes and the position of helix C. In the presence of an ATP analog only one conformation is observed, indicating that this is the active conformation.

Tyramine fragment binding to BACE-1

Fragment screening revealed that tyramine binds to the active site of the Alzheimers disease drug target BACE-1. Hit expansion by selection of compounds from the Roche compound library identified tyramine derivatives with improved binding affinities as monitored by surface plasmon resonance. X-ray structures show that the amine of the tyramine fragment hydrogen-bonds to the catalytic water molecule. Structure-guided ligand design led to the synthesis of further low molecular weight compounds that are starting points for chemical leads.

Structure-Based Drug Design of RN486, a Potent and Selective Bruton's Tyrosine Kinase (BTK) Inhibitor, for the Treatment of Rheumatoid Arthritis.

Structure-based drug design was used to guide the optimization of a series of selective BTK inhibitors as potential treatments for Rheumatoid arthritis. Highlights include the introduction of a benzyl alcohol group and a fluorine substitution, each of which resulted in over 10-fold increase in activity. Concurrent optimization of drug-like properties led to compound 1 (RN486) ( J. Pharmacol. Exp. Ther. 2012 , 341 , 90 ), which was selected for advanced preclinical characterization based on its favorable properties.

Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures.

Brutons tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X‐ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly‐rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2‐kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 310‐ and α‐helices, one of which collapses into the adenosine‐5′‐triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG‐out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure‐based design of specific inhibitors.

Charge separation induces conformational changes in the photosynthetic reaction centre of purple bacteria

X-ray structures of the wild-type reaction centre from Rhodobacter sphaeroides have been determined to a resolution of 1.87 A in the neutral (dark) state and to 2.06 A in the charge-separated (light-excited) state. Whereas the overall protein structures of both states are rather similar, the domain around the secondary quinone shows significant shifts. The quinone molecule itself is observed at two different positions. In the neutral state, 55% of the quinone is located distally and 45% proximally to the cytoplasmic side. After excitation by light, however, at least 90% of the quinone is found at the proximal position. Results presented by Stowell et al. (1997) are confirmed, but the quality of crystallographic data has been improved. We compare the data with the structure of the mutant RC L 209 PY that keeps the Q(B) molecule in the proximal position even in the charge-neutral state.

A Real-World Perspective on Molecular Design.

We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.