Design principles for balanced lipophilicity and permeability in beyond Rule of 5 space

A conformational analysis of all oral bRo5 drugs using a QM-based workflow and experimental structures revealed similar polar surface area (PSA) thresholds as for Ro5 drugs and a modest impact of environments on 3D-PSA and intramolecular hydrogen bond (IMHB) count (often termed chameleonicity) despite a significant difference between TPSA and 3D PSA. The minimum TPSA-3D PSA to maintain permeability in bRo5 space depends on the fraction of polarity (TPSA/MW). TPSA/MW in the bRo5 and Ro5 oral drugs sets had a median of ~0.2 Å2/Da, with the upper half corresponding to the upper decile of logP 100 Å2 de-fines the sweet spot of this "rule of 1/5" occupied by the majority of oral bRo5 drugs. TPSA-3DPSA increased in the lead optimization (LO) campaigns of three first in class de novo designed bRo5 drugs and may be a useful parameter for future bRo5 LO campaigns

The ABC of protein kinase conformations

Conformation plays a crucial role in kinase biology. In this paper, we present a ternary classification scheme of the structural kinome based on the conformation of the DFG-motif (DFG-F and DFG-G side chain torsion) and the displacement of aC-helix and DFG, revealing a small set of major conformations. The DFG-motif occurs in three eclipsed conformations which differ in propensity for aC-helix displacement. The tight interaction between aC-helix and DFG forms a strong bias towards the active conformation and organizes the universal features of active kinases. The activating effect of sequence, phosphorylation, cyclin and inhibitor binding can be estimated by thermodynamic analyses. A likely path for the DFG-out transition, rotation around the bond between DFG-1 and DFG-F after a backbone flip, is suggested by its population in the pdb. Consistent with this mechanism, flexible, lipophilic residues in the path of the DFG-F sidechain and flexibility of aC-helix are required for the transition. Displacement of aC-helix involves subtle shifts in improper torsion angles which propagate to a bending of the C-terminal loop between aC-helix and the HPN motif

The 10 things you should know about protein kinases and inhibitors

Many human malignancies are associated with deregulated activation of protein or lipid kinases due to mutations, chromosomal rearrangements and/or gene amplification. Protein and lipid kinases, therefore, represent, after GPCRs and proteases an important target classes for treating human disorders. This review focus on “the 10 things you should know about protein kinases and their inhibitors” including a short introduction on the history on protein kinases and inhibitor and ending with a perspective in kinase drug discovery. Although the “10 things” have been, to a certain extent, chosen arbitrarily, they cover in a comprehensive way the past and present efforts in kinase drug discovery and summarize the “status quo” of the current kinase inhibitors as well as knowledge about kinase structure and binding modes. Besides describing the potentials of protein kinase inhibitors as drugs this review also focus on their limitations, in particular on how to circumvent emerging resistance against kinase inhibitors in oncological indications

Expanding the Opportunities for Modulating Kinase Targets with Allosteric Approaches

The need for novel approaches for targeting well-known protein families in drug discovery has been discussed for several years. There is a huge amount of literature on the inhibition of kinases with small molecules targeting the ATP site, and as a result of this extensive research, there are a large number of kinase inhibitors in the clinic. However, even though the idea of targeting other sites on kinases is not new, relatively little has been reported. In this review we give an overview of structurally characterized allosteric kinase inhibitors, outline the benefits of these with the use of case studies and then discuss the challenges that need to be overcome and the opportunities for doing this

Type II inhibitors targeting CDK2

Kinases can switch between active and inactive conformations of the ATP/Mg2+ binding motif DFG motif which has been explored by the development of type I or type II inhibitors. However, factors modulating DFG conformational remain poorly understood. We chose CDK2 as a model system to study the DFG out transition on a target that was thought to have an inaccessible DFG-out conformation using site directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. 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 a aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II CDK2 co-crystal 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 dissociation 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 provide also 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

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

Mutational analysis of isoform selectivity and conformational equilibria in protein kinase inhibition

Deregulation of protein kinases is associated with many diseases making them important targets for therapeutic intervention. Kinases can switch between active and inactive conformations that can be targeted by type 1 or type 2 inhibitors respectively. One of the most relevant conformational switches is the ‘in’ and ‘out’ movement of the ATP/Mg2+ binding motif DFG. Factors modulating the conformational equilibria such as the residue environment of regulatory motifs remain poorly understood despite their importance for drug discovery. In this thesis, the first model system tested the hypothesis that accessibility of the DFG-out conformation is restricted by the energetic cost of transition between the in and out states. CDK2 was chosen as a target that was thought to have an inaccessible DFG-out conformation, and several point mutations were introduced to promote this conformational transition. Detailed biochemical and biophysical characterisation illustrated that the mutants bound type 2 inhibitors more potently than the wild type. In addition, the wild-type CDK2 was shown to bind type 2 inhibitors in the absence, but not in the presence, of cyclin. The first known CDK2 co-crystal structure in the DFG-out conformation was solved, opening the door to a new class of CDK2 inhibitors. In the second project, site-directed mutagenesis was used to explore the residues determining inhibitor selectivity between PIM1 and PIM2. Evaluation of ligand binding to the variants and comparison of PIM1 and PIM2 crystal structures showed that flexibility of the phosphate-binding loop was the dominant factor determining the differences in their affinities for ATP and small molecule inhibitors. These studies illustrate that residues contributing to kinase conformational equilibria can be just as important for inhibitor binding as contact residues formed in the ligand complex.This thesis is not currently available in ORA

The Substrate-Activity-Screening methodology applied to receptor tyrosine kinases: A proof-of-concept study.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. A wide variety of disorders can arise as a consequence of abnormal kinase-mediated phosphorylation and numerous kinase inhibitors have earned their place as key components of the modern pharmacopeia. Although “traditional” kinase inhibitors typically act by preventing the interaction between the kinase and ATP, thus stopping substrate phosphorylation, an alternative approach consists in disrupting the protein-protein interaction between the kinase and its downstream partners. We herein report a proof-of-concept application of the Substrate Activity Screening methodology to Insulin-like Growth Factor 1 Receptor as a tool for the discovery of substrate-site-directed tyrosine kinase inhibitors

Substrate profiling of IGF-1R and InsR: Identification of a potent pentamer substrate.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. As part of a study on the substrate requirements of Insulin-like Growth Factor 1 Receptor (IGF-1R) and Insulin Receptor (InsR), we evaluated and applied a universal assay system able to monitor the phosphorylation of unlabelled peptides of any length in real time. In contrast to already reported profiling methodologies, we were able to assess the kcat/KM ratio of peptides as short as tetramers. Notably, we were able to identify an efficient pentamer substrate that exhibited kinetic properties close to those of a 250-amino acid protein derived from IRS-1, a natural substrate of IGF-1R and InsR

Optimization of a fragment-based screening hit towards 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 hypermeth-ylation of H3K79 and misexpression of genes (including HoxA) which drive the leukemic phenotype. A weak fragment-based screening hit identified by SPR was co-crystallized 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 with the SAM binding pocket but induces a pocket adjacent to the SAM binding site