Structural and energetic insights into the selective inhibition of PKMYT1 against WEE1.

Protein kinase, membrane-associated tyrosine/threonine 1 (PKMYT1), a member of the WEE family and responsible for the regulation of CDK1 phosphorylation, has been considered a promising therapeutic target for cancer therapy. However, the highly structural conservation of the ATP-binding sites of the WEE family poses a challenge to the design of selective inhibitors for PKMYT1. Here, molecular docking, multiple microsecond-length molecular dynamics (MD) simulations and end-point free energy calculations were performed to uncover the molecular mechanism of the binding selectivity of RP-6306 toward PKMYT1 over its highly homologous kinase WEE1. The binding specificity of RP-6306 reported in previous experimental bioassays was clarified by MD simulations and binding free energy calculations. Further, the binding free energy prediction indicated that the binding selectivity of RP-6306 largely derived from the difference in the protein-ligand electrostatic interactions. The per-residue free energy decomposition suggested that the non-conserved gatekeeper residue in the hinge domain of PKMYT1/WEE1, Thr187/Asn376, is the critical factor responsible for the binding selectivity of RP-6306 toward PKMYT1. In addition, a water-mediated hydrogen bond was formed between RP-6306 and Gly191 at the hinge domain in the PKMYT1/RP-6306 complex, which was absent in the WEE1/RP-6306 complex. This study is expected to offer useful information for the design of more potent and selective PKMYT1 inhibitors.Communicated by Ramaswamy H. Sarma.

Prognostic Value of miR-1826 in Prostate Cancer and Its Regulatory Effect on Tumor Progression.

PURPOSE: miRNAs can act as oncogenes or tumor suppressors and participate in the development and progression of tumors, thus affecting the prognosis and survival of cancer patients. In this paper, we mainly studied the role of miR-1826 in prostate cancer. PATIENTS AND METHODS: The expression of miR-1826 was studied by quantitative real-time PCR (qRT-PCR). Kaplan-Meier curves were used to analyze the relationship between the expression of miR-1826 and the survival rate of PC patients. Cox regression analysis was used to study the risk factors affecting the prognosis of PC patients. PC cells were transfected with miR-1826 mimic, mimic negative control (mimic NC), miR-1826 inhibitor, or inhibitor NC. The effect of miR-1826 on the proliferation of PC cells was studied by the CCK-8 method and colony formation assay. Transwell assays were used to detect the effect of miR-1826 on the migratory and invasive abilities of tumor cells. RESULTS: The expression of miR-1826 in PC tissues was lower than that in adjacent normal tissues, and that the expression levels of miR-1826 in four PC cell lines were all lower than normal human prostate epithelial cell lines. Patients with low expression of miR-1826 had shorter overall survival compared with those with high expression. The downregulation of miR-1826 promoted PC cell proliferation, migration, and invasion. CONCLUSION: In summary, the low expression of miR-1826 may promote the progression of PC, and the low expression of miR-1826 is also associated with a poor prognosis in PC patients.

LINC-PINT Inhibited Malignant Progression of Bladder Cancer by Targeting miR-155-5p.

BACKGROUND: This study mainly explored the expression level of LINC-PINT in bladder cancer and its relationship with prognosis. Meanwhile, the effect of LINC-PINT on the biological function of bladder cancer was also explored. METHODS: The expression levels of LINC-PINT and miR-155-5p were detected by qRT-PCR. The prognostic significance of LINC-PINT in bladder cancer was studied by the Kaplan-Meier curve and Log rank test. CCK-8 and Transwell assays were used to analyze the proliferation, migration, and invasion ability. The targeting relationship between LINC-PINT and miR-155-5p was analyzed using bioinformatics and dual-luciferase reporter assays. RESULTS: The expression of LINC-PINT was downregulated in bladder cancer tissues and cell lines, and miR-155-5p showed the opposite trend in bladder cancer tissues. Kaplan-Meier curve proved that the patients with low LINC-PINT expression had a lower five-year survival rate and the Log rank test displayed that LINC-PINT was a prognostic factor of BC. CCK-8 and Transwell results showed that LINC-PINT could inhibit the ability of proliferation, migration, and invasion. LINC-PINT was proved to target miR-155-5p in bladder cancer. Dual-luciferase reporter gene assay showed that the relative luciferase activity of overexpression miR-155-5p co-transfected with LINC-PINT-wt was significantly lower. LINC-PINT was negatively correlated with miR-155-5p. CONCLUSION: LINC-PINT is a potential prognostic marker of bladder cancer, and the up-regulation of Lin-PINT can inhibit the proliferation, invasion, and migration of bladder cancer cells by targeting miR-155-5p.

Deciphering the Mechanism of Gilteritinib Overcoming Lorlatinib Resistance to the Double Mutant I1171N/F1174I in Anaplastic Lymphoma Kinase.

Anaplastic lymphoma kinase (ALK) is validated as a therapeutic molecular target in multiple malignancies, such as non-small cell lung cancer (NSCLC). However, the feasibility of targeted therapies exerted by ALK inhibitors is inevitably hindered owing to drug resistance. The emergence of clinically acquired drug mutations has become a major challenge to targeted therapies and personalized medicines. Thus, elucidating the mechanism of resistance to ALK inhibitors is helpful for providing new therapeutic strategies for the design of next-generation drug. Here, we used molecular docking and multiple molecular dynamics simulations combined with correlated and energetical analyses to explore the mechanism of how gilteritinib overcomes lorlatinib resistance to the double mutant ALK I1171N/F1174I. We found that the conformational dynamics of the ALK kinase domain was reduced by the double mutations I1171N/F1174I. Moreover, energetical and structural analyses implied that the double mutations largely disturbed the conserved hydrogen bonding interactions from the hinge residues Glu1197 and Met1199 in the lorlatinib-bound state, whereas they had no discernible adverse impact on the binding affinity and stability of gilteritinib-bound state. These discrepancies created the capacity of the double mutant ALK I1171N/F1174I to confer drug resistance to lorlatinib. Our result anticipates to provide a mechanistic insight into the mechanism of drug resistance induced by ALK I1171N/F1174I that are resistant to lorlatinib treatment in NSCLC.

The structural basis of the autoinhibition mechanism of glycogen synthase kinase 3ß (GSK3ß): molecular modeling and molecular dynamics simulation studies.

The autoinhibition phenomenon has been frequently observed in enzymes and represents an important regulatory strategy to fine-tune enzyme activity. Evolution has exploited this mechanism to reduce enzymatic activity. Glycogen synthase kinase 3ß (GSK3ß) undergoes autoinhibition via the phosphorylation of Ser9 at the N-terminus of the kinase, which, acting as a pseudosubstrate, occupies the catalytic domain of GSK3ß and subsequently blocks primed substrates from having access to the catalytic domain. The detailed structural basis of the autoinhibition mechanism of GSK3ß by the pseudosubstrate, however, has not yet been fully resolved. Here, a three-dimensional model of the binary GSK3ß-pseudosubstrate complex was built via the molecular modeling method. Based on the constructed model, extensive molecular dynamics (MD) simulations and subsequent molecular mechanics generalized Born/surface area (MM_GBSA) calculations were performed on the wild-type GSK3ß-pseudosubstrate complex and three mutated systems (R4A, R6A, and S9A). Analyses of MD simulations and binding free energies revealed that the phosphorylation of Ser9 is the prerequisite for the autoinhibition of GSK3ß, and both mutations of Arg4 and Arg6 to alanine markedly reduced the binding affinities of the mutated pseudosubstrate to the GSK3ß catalytic domain, thereby disrupting the autoinhibition of the kinase. This study highlights the importance of Ser9, Arg6, and Arg4 in modulating the autoinhibition mechanism of GSK3ß, contributing to a deeper understanding of GSK3ß biology.Communicated by Ramaswamy H. Sarma.

How does the novel T315L mutation of breakpoint cluster region-abelson (BCR-ABL) kinase confer resistance to ponatinib: a comparative molecular dynamics simulation study.

Acute lymphocytic leukemia (ALL) is one of the most dangerous types of leukemia, and about 40% of them is Philadelphia chromosome-positive acute lymphocytic leukemia (Ph + ALL). Ph + ALL is caused by the fusion of the breakpoint cluster region (BCR) and the Ableson (ABL) genes, named the BCR-ABL fused gene that codes for an autonomously active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are among the first-line therapeutic agents for the treatment of Ph + ALL. Drug resistance are the major obstacle, limiting their clinical utility. The latest third-generation TKIs, ponatinib, can tackle most abnormal BCR-ABL kinases, including the T315I mutant that is resistant to first- and second-generations TKIs such as imatinib. However, drug resistance still emerges with the novel T315L mutation and the underlying mechanisms remain elusive. Here, using molecular dynamics (MD) simulations, we explored into the detailed interactions between ponatinib and BCR-ABL in the wild-type (WT), T315I, and T315L systems. The simulations revealed the significant conformational changes of ponatinib in its binding site due to the T315L mutation and the underlying structural mechanisms. Binding free energy analysis unveiled that the affinity of ponatinib to BCR-ABL decreased upon T315L mutation, which resulted in its unfavorable binding and drug resistance. Key residues responsible for the unfavored unbinding were also identified. This study elucidates the detailed mechanisms for the resistance of ponatinib in Ph + ALL triggered by the T315L mutation and will provide insights for future drug development and optimization.

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 µs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity.

Unraveling the role of Arg4 and Arg6 in the auto-inhibition mechanism of GSK3ß from molecular dynamics simulation.

Glycogen synthase kinase 3ß (GSK3ß) is a multifunctional serine/threonine protein kinase that is involved in several biological processes including insulin and Wnt signaling pathways. GSK3ß can be phosphorylated by the protein kinase B (PKB). The mutations of Arg4 and Arg6 to alanine at N-terminal GSK3ß have been reported to impair its ability to autophosphorylate at Ser9. Despite the extensive experimental observations, the detailed mechanism for the auto-inhibition of GSK3ß has not been rationalized at the molecular level. In this study, we have demonstrated the structural consequences of GSK3ß R4A and R6A mutations and the atomic changes that influenced the loss of PKB-binding affinity. Molecular dynamics simulation results suggested significant loss in atomic contacts in the R4A and R6A mutant systems compared to the wild-type system. Furthermore, we observed many notable changes (such as conformation, residues motions, hydrogen bonds, and binding free energy) in the mutated GSK3ß-PKB complexes. Loss of binding affinity in the mutated systems rendered the decrease in GSK3ß phosphorylation, which, in turn, impaired the auto-inhibition of GSK3ß. The significant outcomes obtained from this study can explain the auto-inhibition of GSK3ß and maybe facilitate type 2 diabetes mellitus researches and in developing the potent drug therapies.

ASD v2.0: updated content and novel features focusing on allosteric regulation.

Allostery is the most direct and efficient way for regulation of biological macromolecule function and is induced by the binding of a ligand at an allosteric site topographically distinct from the orthosteric site. AlloSteric Database (ASD, http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information on allostery. Owing to the inherent high receptor selectivity and lower target-based toxicity, allosteric regulation is expected to assume a more prominent role in drug discovery and bioengineering, leading to the rapid growth of allosteric findings. In this updated version, ASD v2.0 has expanded to 1286 allosteric proteins, 565 allosteric diseases and 22 008 allosteric modulators. A total of 907 allosteric site-modulator structural complexes and >200 structural pairs of orthosteric/allosteric sites in the allosteric proteins were constructed for researchers to develop allosteric site and pathway tools in response to community demands. Up-to-date allosteric pathways were manually curated in the updated version. In addition, both the front-end and the back-end of ASD have been redesigned and enhanced to allow more efficient access. Taken together, these updates are useful for facilitating the investigation of allosteric mechanisms, allosteric target identification and allosteric drug discovery.

Allosite: a method for predicting allosteric sites.

MOTIVATION: The use of allosteric modulators as preferred therapeutic agents against classic orthosteric ligands has colossal advantages, including higher specificity, fewer side effects and lower toxicity. Therefore, the computational prediction of allosteric sites in proteins is receiving increased attention in the field of drug discovery. Allosite is a newly developed automatic tool for the prediction of allosteric sites in proteins of interest and is now available through a web server. AVAILABILITY: The Allosite server and tutorials are freely available at http://mdl.shsmu.edu.cn/AST CONTACT: jian.zhang@sjtu.edu.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.