Thierry O. Fischmann

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 Å resolution) and iNOS (2.25 Å resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

Dinaciclib (SCH 727965), a Novel and Potent Cyclin-Dependent Kinase Inhibitor

Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC50 values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters. Compared with flavopiridol, SCH 727965 exhibits superior activity with an improved therapeutic index. In cell-based assays, SCH 727965 completely suppressed retinoblastoma phosphorylation, which correlated with apoptosis onset and total inhibition of bromodeoxyuridine incorporation in >100 tumor cell lines of diverse origin and background. Moreover, short exposures to SCH 727965 were sufficient for long-lasting cellular effects. SCH 727965 induced regression of established solid tumors in a range of mouse models following intermittent scheduling of doses below the maximally tolerated level. This was associated with modulation of pharmacodynamic biomarkers in skin punch biopsies and rapidly reversible, mechanism-based effects on hematologic parameters. These results suggest that SCH 727965 is a potent and selective CDK inhibitor and a novel cytotoxic agent. Mol Cancer Ther; 9(8); 2344–53. ©2010 AACR.

Selective small-molecule inhibition of an RNA structural element.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

Crystal Structures of MEK1 Binary and Ternary Complexes with Nucleotides and Inhibitors.

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35-382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-gammaS determined at 2.1 A. Unlike crystals of a truncated kinase domain previously published, the crystals of the intact domain can be grown either as a binary complex with a nucleotide or as a ternary complex with a nucleotide and one of a multitude of allosteric inhibitors. Further, the crystals allow for the determination of costructures with ATP competitive inhibitors. We describe the structures of nonphosphorylated MEK1 (npMEK1) binary complexes with ADP and K252a, an ATP-competitive inhibitor (see Table 1), at 1.9 and 2.7 A resolution, respectively. Ternary complexes have also been solved between npMEK1, a nucleotide, and an allosteric non-ATP competitive inhibitor: ATP-gammaS with compound 1 and ADP with either U0126 or the MEK1 clinical candidate PD325089 at 1.8, 2.0, and 2.5 A, respectively. Compound 1 is structurally similar to PD325901. These structures illustrate fundamental differences among various mechanisms of inhibition at the molecular level. Residues 44-51 have previously been shown to play a negative regulatory role in MEK1 activity. The crystal structure of the integral kinase domain provides a structural rationale for the role of these residues. They form helix A and repress enzymatic activity by stabilizing an inactive conformation in which helix C is displaced from its active state position. Finally, the structure provides for the first time a molecular rationale that explains how mutations in MEK may lead to the cardio-facio-cutaneous syndrome.

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Structural basis of CX-4945 binding to human protein kinase CK2.

Protein kinase CK2 (CK2), a constitutively active serine/threonine kinase, is involved in a variety of roles essential to the maintenance of cellular homeostasis. Elevated levels of CK2 expression results in the dysregulation of key signaling pathways that regulate transcription, and has been implicated in cancer. The adenosine‐5′‐triphosphate‐competitive inhibitor CX‐4945 has been reported to show broad spectrum anti‐proliferative activity in multiple cancer cell lines. Although the enzymatic IC50 of CX‐4945 has been reported, the thermodynamics and structural basis of binding to CK2α remained elusive. Presented here are the crystal structures of human CK2α in complex with CX‐4945 and adenylyl phosphoramidate at 2.7 and 1.3 Å, respectively. Biophysical analysis of CX‐4945 binding is also described. This data provides the structural rationale for the design of more potent inhibitors against this emerging cancer target.

Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2.

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.

Design, synthesis and SAR of thienopyridines as potent CHK1 inhibitors.

A novel series of CHK1 inhibitors based on thienopyridine template has been designed and synthesized. These inhibitors maintain critical hydrogen bonding with the hinge and conserved water in the ATP binding site. Several compounds show single digit nanomolar CHK1 activities. Compound 70 shows excellent enzymatic activity of 1 nM.

Discovery of a Novel Series of CHK1 Kinase Inhibitors with a Distinctive Hinge Binding Mode.

A novel series of CHK1 inhibitors with a distinctive hinge binding mode, exemplified by 2-aryl-N-(2-(piperazin-1-yl)phenyl)thiazole-4-carboxamide, was discovered through high-throughput screening using the affinity selection-mass spectrometry (AS-MS)-based Automated Ligand Identification System (ALIS) platform. Structure-based ligand design and optimization led to significant improvements in potency to the single digit nanomolar range and hundred-fold selectivity against CDK2.

Potent and Selective Amidopyrazole Inhibitors of IRAK4 That Are Efficacious in a Rodent Model of Inflammation

IRAK4 is a critical upstream kinase in the IL-1R/TLR signaling pathway. Inhibition of IRAK4 is hypothesized to be beneficial in the treatment of autoimmune related disorders. A screening campaign identified a pyrazole class of IRAK4 inhibitors that were determined by X-ray crystallography to exhibit an unusual binding mode. SAR efforts focused on the identification of a potent and selective inhibitor with good aqueous solubility and rodent pharmacokinetics. Pyrazole C-3 piperidines were well tolerated, with N-sulfonyl analogues generally having good rodent oral exposure but poor solubility. N-Alkyl piperidines exhibited excellent solubility and reduced exposure. Pyrazoles possessing N-1 pyridine and fluorophenyl substituents were among the most active. Piperazine 32 was a potent enzyme inhibitor with good cellular activity. Compound 32 reduced the in vivo production of proinflammatory cytokines and was orally efficacious in a mouse antibody induced arthritis disease model of inflammation.