Henrik Möbitz

Targeting Cancer with Small-Molecular-Weight Kinase Inhibitors

Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions. Deregulation of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. 518 protein kinases and about 20 lipid-modifying kinases are encoded by the human genome, and a much larger proportion of additional kinases are present in parasite, bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry. Approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases.The kinase inhibitors that have been launched, thus far, are mainly in oncology indications and are directed against a handful of protein and lipid kinases. With one exception, all of these registered kinase inhibitors are directed toward the ATP-site and display different selectivities, potencies, and pharmacokinetic properties. At present, about 150 kinase-targeted drugs are in clinical development and many more in various stages of preclinical development. Kinase inhibitor drugs that are in clinical trials target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger-dependent lipid and protein kinases, and protein kinases that regulate the cell cycle.This review provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.

Discovery of Cyclic Sulfone Hydroxyethylamines as Potent and Selective β-Site APP-Cleaving Enzyme 1 (BACE1) Inhibitors: Structure-Based Design and in Vivo Reduction of Amyloid β-Peptides

Structure-based design of a series of cyclic hydroxyethylamine BACE1 inhibitors allowed the rational incorporation of prime- and nonprime-side fragments to a central core template without any amide functionality. The core scaffold selection and the structure-activity relationship development were supported by molecular modeling studies and by X-ray analysis of BACE1 complexes with various ligands to expedite the optimization of the series. The direct extension from P1-aryl- and heteroaryl moieties into the S3 binding pocket allowed the enhancement of potency and selectivity over cathepsin D. Restraining the design and synthesis of compounds to a physicochemical property space consistent with central nervous system drugs led to inhibitors with improved blood-brain barrier permeability. Guided by structure-based optimization, we were able to obtain highly potent compounds such as 60p with enzymatic and cellular IC(50) values of 2 and 50 nM, respectively, and with >200-fold selectivity over cathepsin D. Pharmacodynamic studies in APP51/16 transgenic mice at oral doses of 180 μmol/kg demonstrated significant reduction of brain Aβ levels.

Structural biology contributions to tyrosine kinase drug discovery

Successful kinase inhibitor drug discovery relies heavily on the structural knowledge of the interaction of inhibitors with the target. Structural biology of kinases and in particular of tyrosine kinases has given detailed insights into the intrinsic flexibility of the catalytic domain and has provided a rational basis for obtaining selective inhibitors. Important progress has been made recently, both in academia and in the pharmaceutical industry, with respect to solving structures of inactive, multidomain or protein-protein complexes of kinases, which helps our understanding of the dynamics of regulation of kinase activity. This leads to a better understanding of how mutations lead to activation of kinases and resistance, in addition to providing opportunities for novel modes of targeting kinases.

Structure based design, synthesis and SAR of cyclic hydroxyethylamine (HEA) BACE-1 inhibitors.

This Letter describes the de novo design of non-peptidic hydroxyethylamine (HEA) inhibitors of BACE-1 by elimination of P-gp contributing amide attachments. The predicted binding mode of the novel cyclic sulfone HEA core template was confirmed in a X-ray co-crystal structure. Inhibitors of sub-micromolar potency with an improved property profile over historic HEA inhibitors resulting in improved brain penetration are described.

In vivo and in vitro SAR of tetracyclic MAPKAP-K2 (MK2) inhibitors. Part II.

Spirocyclopropane- and spiroazetidine-substituted tetracycles 13D-E and 16A are described as orally active MK2 inhibitors. The spiroazetidine derivatives are potent MK2 inhibitors with IC(50)<3 nM and inhibit the release of TNFalpha (IC(50)<0.3 microM) from hPBMCs and hsp27 phosphorylation in anisomycin stimulated THP-1 cells. The spirocyclopropane analogues are less potent against MK2 (IC(50)=0.05-0.23 microM), less potent in cells (IC(50)<1.1 microM), but show good oral absorption. Compound 13E (100mg/kg po; bid) showed oral activity in rAIA and mCIA, with significant reduction of swelling and histological score.

The ABC of protein kinase conformations

Due to their involvement in human diseases, protein kinases are an important therapeutic target class. Conformation is a key concept for understanding how functional activity, inhibition and sequence are linked. We assemble and annotate the mammalian structural kinome from the Protein Data Bank on the basis of a universal residue nomenclature. We identify a torsion angle around the Gly of the DFG-motif whose sharp distribution profile corresponds to three eclipsed conformations. This allows the definition a small set of clusters whose distribution shows a bias for the active conformation. A common rationale links the active and inactive state: stabilization of the active conformation, as well as inactivation by displacement of helix-αC or the DFG-motif is governed by the interaction between helix-αC and the DFG motif. In particular, the conformation of the DFG-motif is tightly correlated with the propensity of helix-αC displacement. Our analysis reveals detailed mechanisms for the displacement of helix-αC and the DFG and improves our understanding of the role of individual residues. By pooling conformations from the whole structural kinome, the energetic contributions of sequence and extrinsic factors can be estimated in free energy analyses. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor.

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase.

Type II Inhibitors Targeting CDK2

Kinases can switch between active and inactive conformations of the ATP/Mg(2+) binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in-out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target.

Discovery of Novel Dot1L Inhibitors through a Structure-Based Fragmentation Approach.

Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to repression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket binding mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morphing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors.

Optimization of a Fragment-Based Screening Hit toward Potent DOT1L Inhibitors Interacting in an Induced Binding Pocket.

Mixed lineage leukemia (MLL) gene rearrangement induces leukemic transformation by ectopic recruitment of disruptor of telomeric silencing 1-like protein (DOT1L), a lysine histone methyltransferase, leading to local hypermethylation of H3K79 and misexpression of genes (including HoxA), which drive the leukemic phenotype. A weak fragment-based screening hit identified by SPR was cocrystallized with DOT1L and optimized using structure-based ligand optimization to yield compound 8 (IC50 = 14 nM). This series of inhibitors is structurally not related to cofactor SAM and is not interacting within the SAM binding pocket but induces a pocket adjacent to the SAM binding site.