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 <i>K</i>_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

Virtual Screening of PRK1 Inhibitors: Ensemble Docking, Rescoring Using Binding Free Energy Calculation and QSAR Model Development

Protein kinase C Related Kinase 1 (PRK1) has been shown to be involved in the regulation of androgen receptor signaling and has been identified as a novel potential drug target for prostate cancer therapy. Since there is no PRK1 crystal structure available to date, multiple PRK1 homology models were generated in order to address the protein flexibility. An in-house library of compounds tested on PRK1 was docked into the ATP binding site of the generated models. In most cases a correct pose of the inhibitors could be identified by ensemble docking, while there is still a challenge of finding a reasonable scoring function that is able to rank compounds according to their biological activity. We estimated the binding free energy for our data set of structurally diverse PRK1 inhibitors using the MM-PB­(GB)­SA and QM/MM-GBSA methods. The obtained results demonstrate that a correlation between calculated binding free energies and experimental IC₅₀ values was found to be usually higher than using docking scores. Furthermore, the developed approach was tested on a set of diverse PRK1 inhibitors taken from literature, which resulted in a significant correlation. The developed method is computationally inexpensive and can be applied as a postdocking filter in virtual screening as well as for optimization of PRK1 inhibitors in order to prioritize compounds for further biological characterization

Known and newly identified PRK1 inhibitors.

<p>Known and newly identified PRK1 inhibitors.</p

PRK1 in vitro binding.

<p>PRK1 in vitro binding.</p

Effects of lestaurtinib on the mRNA expression of the androgen receptor target genes.

<p>Expression of androgen receptor target genes <i>TMPRSS2</i>, <i>IGF1-R</i>, <i>NKX3.1</i>, <i>CXCR4</i>, <i>MAK</i>, <i>MAF</i>, <i>N4A1</i>, <i>GREB1</i> and <i>FKBP5</i> measured by qRT-PCR in androgen (R1881) stimulated LNCaP prostate cancer cells are lowered by the treatment with lestaurtinib (final concentration 5 µM). The mRNA expression of the <i>GAPDH</i> gene was used as a control. Bars represent mean + SD (n = 5). P-value: ns  =  non significant; *  =  <0.05; **<0.01; ***<0.001.</p

Details of the binding of (A) staurosporine (green), (B) K252a (orange), (C) lestaurtinib (cyan), and (D) Ro318220 (dark-yellow) to the PRK1 kinase domain.

<p>Common interactions of the inhibitors are hydrogen bonds involving Glu696 and Ser698 of the hinge region, and van-der-Waals interactions with the gatekeeper residue Met695, as well as with Val629, Phe626, Leu747, and Phe904. In addition, some of the inhibitors interact with Asp744, Asn745 and a conserved water molecule (red sphere) nearby the Mg²⁺ binding site of the kinase. The backbone is shown as a purple ribbon. Only relevant amino acids are displayed. Hydrogen bonds are shown as dashed orange colored lines.</p