Identification of PKMYT1 inhibitors by screening the GSK published protein kinase inhibitor set I and II.

As a member of the Wee-kinase family protein kinase PKMYT1 is involved in G2/M checkpoint regulation of the cell cycle. Recently, a peptide microarray approach led to the identification of a small peptide; EFS247-259 as substrate of PKMYT1, which allowed for subsequent development of an activity assay. The developed activity assay was used to characterize the PKMYT1 catalyzed phosphorylation of EFS247-259. For the first time kinetic parameters for PKMYT1, namely Km, Km, ATP and vmax were determined. The optimized assay was used to screen the published protein kinase inhibitor sets (PKIS I and II), two sets of small molecule ATP-competitive kinase inhibitors reported by GlaxoSmithKline. We identified ten inhibitors, providing different scaffolds. The inhibitors were further characterized by using binding assay, activity and functional assay. In addition, docking studies were carried out in order to rationalize the observed biological activities. The derived results provide the basis for further chemical optimization of PKMYT1 inhibitors and for further analysis of PKMYT1 as target for anti-cancer therapy.

Regulation of G2/M Transition by Inhibition of WEE1 and PKMYT1 Kinases.

In the cell cycle, there are two checkpoint arrests that allow cells to repair damaged DNA in order to maintain genomic integrity. Many cancer cells have defective G1 checkpoint mechanisms, thus depending on the G2 checkpoint far more than normal cells. G2 checkpoint abrogation is therefore a promising concept to preferably damage cancerous cells over normal cells. The main factor influencing the decision to enter mitosis is a complex composed of Cdk1 and cyclin B. Cdk1/CycB is regulated by various feedback mechanisms, in particular inhibitory phosphorylations at Thr14 and Tyr15 of Cdk1. In fact, Cdk1/CycB activity is restricted by the balance between WEE family kinases and Cdc25 phosphatases. The WEE kinase family consists of three proteins: WEE1, PKMYT1, and the less important WEE1B. WEE1 exclusively mediates phosphorylation at Tyr15, whereas PKMYT1 is dual-specific for Tyr15 as well as Thr14. Inhibition by a small molecule inhibitor is therefore proposed to be a promising option since WEE kinases bind Cdk1, altering equilibria and thus affecting G2/M transition.

Identification of peptidic substrates for the human kinase Myt1 using peptide microarrays.

Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling.

Application of docking and QM/MM-GBSA rescoring to screen for novel Myt1 kinase inhibitors.

Identification of compounds that can bind to a target protein with high affinity is a nontrivial task in structure-based drug design. Several approaches ranging from simple scoring methods to more computationally demanding methods are usually applied for this purpose. In the current work, we used ligand docking in combination with QM/MM-GBSA, MM-GBSA, and MM-PBSA rescoring to discriminate between active and inactive Myt1 kinase inhibitors. Results show that QM/MM-GBSA rescoring performs better than normal docking scores or MM-GBSA rescoring in classifying active and inactive inhibitors. We also applied QM/MM-GBSA rescoring to estimate the binding affinities of compounds from different virtual screening runs. To prove our approach and to confirm its predictive power, a few compounds which were predicted to be active were purchased and experimentally tested. Among the five selected compounds, three showed significant inhibition of recombinant Myt1. PD-173952, which yielded a favorable QM/MM-GBSA binding free energy, showed a K(i) value of 8.1 nM. In addition, two compounds, PD-180970 and saracatinib, showed inhibition at the low micromolar level. Thus, the developed protocol might be useful for further virtual screening experiments to better discriminate between active and inactive compounds and to further optimize the identified hits.

Computer-aided design, synthesis and biological characterization of novel inhibitors for PKMYT1.

In the current work, we applied computational methods to analyze the membrane-associated inhibitory kinase PKMYT1 and small molecule inhibitors. PKMYT1 regulates the cell cycle at G2/M transition and phosphorylates Thr14 and Tyr15 in the Cdk1-cyclin B complex. A combination of in silico and in vitro screening was applied to identify novel PKMYT1 inhibitors. The computational approach combined structural analysis, molecular docking, binding free energy calculations, and quantitative structure-activity relationship (QSAR) models. In addition, a computational fragment growing approach was applied to a set of previously identified diaminopyrimidines. Based on the derived computational models, several derivatives were synthesized and tested in vitro on PKMYT1. Novel inhibitors active in the sub-micromolar range were identified which provide the basis for further characterization of PKMYT1 as putative target for cancer therapy.

Augmented reversible photoswitching of drug-target interaction through "surface borrowing".

Reversibly switching bioactive molecules by light could allow spatiotemporal control of their biological functions, but designing such drug molecules with large functional differences between two light-switchable states remains difficult. We extend the concept of "borrowing protein" to "borrowing surface" for the design of photo-switchable molecules. By using an azobenzene conjugated cyclosporin derivative as a model system we have demonstrated that the enhanced steric hindrance resulting from this "borrowing surface" design can cause augmentation of the functional difference between the cis and trans conformers of this light-switchable compound. Interestingly, not only near UV light of 366nm but also visible light of 430nm or 525nm can induce efficient photoswitching of the interactions between the light-responsive ligand and target protein. Additionally, by using molecular modeling and docking techniques we obtained structural insights into the switchable protein-ligand interaction, illustrating the effect of steric hindrance associated with the borrowing surface design.