Hitomi Yuki

A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo

A pyrrolo-pyrimidine kinase inhibitor, RK-20449, eliminates chemotherapy-resistant human primary AML stem cells in vivo. Taking AML Head-On Like the mythic Lernaean Hydra, acute myeloid leukemia (AML) is hard to kill. It seems like every time one head is cut off, another two—meaner—grow back in its place. Leukemia stem cells (LSCs) are thought to contribute to this resilience; they may survive conventional chemotherapy and increase the risk of relapse. However, it has been difficult to specifically target these cells without also hitting the normal hematopoietic stem cells (HSCs) required for maintaining healthy blood cells. Now, Saito et al. find a new candidate drug that can specifically target LSCs. The authors performed a chemical library screen to target hematopoietic cell kinase (HCK), which they had previously found to be differentially expressed in human LSCs compared with HSCs. They found a candidate HCK inhibitor, RK-20449, which is a pyrrolo-pyrimidine derivative that could bind the active pocket of HCK. In a mouse xenograft of aggressive human AML, RK-20449 greatly reduced LSC burden. If these studies hold true in patients, RK-20449 could accomplish the Herculean task of decreasing the risk of relapse in AML. Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rgnull mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

Mechanism of the decrease in catalytic activity of human cytochrome P450 2C9 polymorphic variants investigated by computational analysis

Cytochrome P450 (CYP) is deeply involved in the metabolism of chemicals including pharmaceuticals. Therefore, polymorphisms of this enzyme have been widely studied to avoid unfavorable side effects of drugs in chemotherapy. In this work, we performed computational analysis of the mechanism of the decrease in enzymatic activity for three typical polymorphisms in CYP 2C9 species: *2, *3, and *5. Based on the equilibrated structure obtained by molecular dynamics simulation, the volume of the binding pocket and the fluctuation of amino residues responsible for substrate holding were compared between the wild type and the three variants. Further docking simulation was carried out to evaluate the appropriateness of the binding pocket to accommodate substrate chemicals. Every polymorphic variant was suggested to be inferior to the wild type in enzymatic ability from the structural viewpoint. F‐G helices were obviously displaced outward in CYP2C9*2. Expansion of the binding pocket, especially the space near F′ helix, was remarkable in CYP2C9*3. Disappearance of the hydrogen bond between K helix and β4 loop was observed in CYP2C9*5. The reduction of catalytic activity of those variants can be explained from the deformation of the binding pocket and the consequent change in binding mode of substrate chemicals. The computational approach is effective for predicting the enzymatic activity of polymorphic variants of CYP. This prediction will be helpful for advanced drug design because calculations forecast unexpected change in drug efficacy for individuals.

Prediction of sites of metabolism in a substrate molecule, instanced by carbamazepine oxidation by CYP3A4.

In drug discovery process, improvement of ADME/Tox properties of lead compounds including metabolic stability is critically important. Cytochrome P450 (CYP) is one of the major metabolizing enzymes and the prediction of sites of metabolism (SOM) on the given lead compounds is key information to modify the compounds to be more stable against metabolism. There are two factors essentially important in SOM prediction. First is accessibility of each substrate atom to the oxygenated Fe atom of heme in a CYP protein, and the other is the oxidative reactivity of each substrate atom. To predict accessibility of substrate atoms to the heme iron, conventional protein-rigid docking simulations have been applied. However, the docking simulations without consideration of protein flexibility often lead to incorrect answers in the case of very flexible proteins such as CYP3A4. In this study, we demonstrated an approach utilizing molecular dynamics (MD) simulation for SOM prediction in which multiple MD runs were executed using different initial structures. We applied this strategy to CYP3A4 and carbamazepine (CBZ) complex. Through 10 ns MD simulations started from five different CYP3A4-CBZ complex models, our approach correctly predicted SOM observed in experiments. The experimentally known epoxidized sites of CBZ by CYP3A4 were successfully predicted as the most accessible sites to the heme iron that was judged from a numerical analysis of calculated ΔG(binding) and the frequency of appearance. In contrast, the predictions using protein-rigid docking methods hardly provided the correct SOM due to protein flexibility or inaccuracy of the scoring functions. Our strategy using MD simulation with multiple initial structures will be one of the reliable methods for SOM prediction.

A novel pim-1 kinase inhibitor targeting residues that bind the substrate Peptide.

A new screening method using fluorescent correlation spectroscopy was developed to select kinase inhibitors that competitively inhibit the binding of a fluorescently labeled substrate peptide. Using the method, among approximately 700 candidate compounds selected by virtual screening, we identified a novel Pim-1 kinase inhibitor targeting its peptide binding residues. X-ray crystal analysis of the complex structure of Pim-1 with the inhibitor indicated that the inhibitor actually binds to the ATP-binding site and also forms direct interactions with residues (Asp128 and Glu171) that bind the substrate peptide. These interactions, which cause small side-chain movements, seem to affect the binding ability of the fluorescently labeled substrate. The compound inhibited Pim-1 kinase in vitro, with an IC(50) value of 150 nM. Treatment of cultured leukemia cells with the compound reduced the amount of p21 and increased the amount of p27, due to Pim-1 inhibition, and then triggered apoptosis after cell-cycle arrest at the G(1)/S phase. This screening method may be widely applicable for the identification of various new Pim-1 kinase inhibitors targeting the residues that bind the substrate peptide.

An epoxidation mechanism of carbamazepine by CYP3A4.

Human CYP3A4 catalyzes the 10,11-epoxidation of carbamazepine (CBZ). However, the epoxide is less stable in terms of potential energy than hydroxides of the six-membered aromatic ring. To clarify the reason why CYP3A4 produces such an energetically unfavorable compound, the mechanism of epoxidation of CBZ by CYP3A4 was investigated by theoretical calculations. The reaction consisted of two elementary processes in which two C-O bonds were generated stepwise. The rate-determining step was the first one and the activation energy was 21.3kcal/mol at the DFT (B3LYP/6-31G( * *)) level. The activation energy level of the first step of the 10,11-epoxidation was lower than that of the hydroxylation of the aromatic ring. For this reason, 10,11-epoxidation is more probable than hydroxylation of the aromatic ring, and only 10,11-epoxide is formed.

Application of support vector machine to three-dimensional shape-based virtual screening using comprehensive three-dimensional molecular shape overlay with known inhibitors.

In this study, machine learning using support vector machine was combined with three-dimensional (3D) molecular shape overlay, to improve the screening efficiency. Since the 3D molecular shape overlay does not use fingerprints or descriptors to compare two compounds, unlike 2D similarity methods, the application of machine learning to a 3D shape-based method has not been extensively investigated. The 3D similarity profile of a compound is defined as the array of 3D shape similarities with multiple known active compounds of the target protein and is used as the explanatory variable of support vector machine. As the measures of 3D shape similarity for our new prediction models, the prediction performances of the 3D shape similarity metrics implemented in ROCS, such as ShapeTanimoto and ScaledColor, were validated, using the known inhibitors of 15 target proteins derived from the ChEMBL database. The learning models based on the 3D similarity profiles stably outperformed the original ROCS when more than 10 known inhibitors were available as the queries. The results demonstrated the advantages of combining machine learning with the 3D similarity profile to process the 3D shape information of plural active compounds.

Implementation of π‐π interactions in molecular dynamics simulation

No explicit π‐π interaction term has been incorporated in the conventional molecular dynamics (MD) simulation programs in spite of its significant role in the folding of biomolecules and the clustering of organic chemicals. In this article, we propose a technique to emphasize the effect of π‐π interactions using a function of energy and implement it into an MD simulation program. Several trial calculations show that the π‐π incorporated program gives improved results consistent with experimental data on atom geometry and has no unfavorable interference with the conventional computational framework. This indicates an importance of the explicit consideration of π‐π interactions in MD simulation.

Insights from Pim1 structure for anti-cancer drug design

Introduction: Pim1 is a unique, constitutively active serine/threonine kinase, and is a potent oncogene overexpressed in a range of hematologic malignancies and solid cancers. It functions as a signaling regulator in cell proliferation and survival pathways through substrate phosphorylation. More than 400 small molecular Pim1 inhibitors with IC50/Ki < 10 μM have been registered in the ChEMBL. However, no drug targeting Pim1 has been launched at this present time. Areas covered: The authors provide a review of Pim1 inhibitors from the viewpoint of the structural analysis of the Pim1-inhibitor complexes. PDB data and PubMed literature searches were performed. The inhibitors have been classified and summarized by their interactions with Pim1 residues, to facilitate desirable inhibitor design. Expert opinion: To obtain a potent and selective Pim1 inhibitor as a lead compound, the authors propose the development of compounds which simultaneously interact with both the ATP binding site (Lys67, Glu121 and Phe49) and substrate binding residues (Asp128, Asp131 and Glu171). The development of Pim1 inhibitors could lead to new therapeutic options for a number of hematological malignancies and prostate cancer in the future.

Flexibility of the P‐loop of Pim‐1 kinase: observation of a novel conformation induced by interaction with an inhibitor

The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012), J. Med. Chem. 55, 5151-5156]. The report described the process of optimization of the structure-activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the β3 strand located above the binding site, is not usually observed in Pim-1 structures.

Identification of novel drug-resistant EGFR mutant inhibitors by in silico screening using comprehensive assessments of protein structures

EGFR is a target protein for the treatment of non small cell lung cancer (NSCLC). The mutations associated with the activation of EGFR kinase activity, such as L858R and G719S, destabilize the inactive conformation of EGFR and are closely linked with the development of NSCLC. The additional T790M mutation reportedly causes drug resistance against the commercially available EGFR inhibitors, gefitinib and erlotinib. In this study, we searched for novel G719S/T790M EGFR inhibitors by a new in silico screening strategy, using two datasets. The results of in silico screening using protein-ligand docking are affected by the selection of 3D structure of the target protein. As the first strategy, we chose the 3D structures for in silico screening by test dockings using the G719S/T790M crystal structure, its molecular dynamics snapshots, and known inhibitors of the drug-resistant EGFR. In the second strategy, we selected the 3D structures by test dockings using all of the EGFR structures, regardless of the mutations, and all of the known EGFR inhibitors. Using each of the 3D structures selected by the strategies, 1000 compounds were chosen from the 71,588 compounds. Kinase assays identified 15 G719S/T790M EGFR inhibitors, including two compounds with novel scaffolds. Analyses of their structure-activity relationships revealed that interactions with the mutated Met790 residue specifically increase the inhibitory activity against G719S/T790M EGFR.