Prediction of Promiscuous P-Glycoprotein Inhibition Using a Novel Machine Learning Scheme

BACKGROUND: P-glycoprotein (P-gp) is an ATP-dependent membrane transporter that plays a pivotal role in eliminating xenobiotics by active extrusion of xenobiotics from the cell. Multidrug resistance (MDR) is highly associated with the over-expression of P-gp by cells, resulting in increased efflux of chemotherapeutical agents and reduction of intracellular drug accumulation. It is of clinical importance to develop a P-gp inhibition predictive model in the process of drug discovery and development. METHODOLOGY/PRINCIPAL FINDINGS: An in silico model was derived to predict the inhibition of P-gp using the newly invented pharmacophore ensemble/support vector machine (PhE/SVM) scheme based on the data compiled from the literature. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those structurally diverse molecules in the training set (n = 31, r(2) = 0.89, q(2) = 0.86, RMSE = 0.40, s = 0.28), the test set (n = 88, r(2) = 0.87, RMSE = 0.39, s = 0.25) and the outlier set (n = 11, r(2) = 0.96, RMSE = 0.10, s = 0.05). The generated PhE/SVM model also showed high accuracy when subjected to those validation criteria generally adopted to gauge the predictivity of a theoretical model. CONCLUSIONS/SIGNIFICANCE: This accurate, fast and robust PhE/SVM model that can take into account the promiscuous nature of P-gp can be applied to predict the P-gp inhibition of structurally diverse compounds that otherwise cannot be done by any other methods in a high-throughput fashion to facilitate drug discovery and development by designing drug candidates with better metabolism profile

Structure-based Design of Small Molecule Inhibitors of HIV-1 Entry

HIV infection begins when gp120 envelope protein on the viral surface binds to the CD4 receptor on the host cell. This initial protein-protein interaction starts the rest of the HIV lifecycle of coreceptor binding, fusion and replication. One of the targets of HIV entry inhibitors is the interaction between CD4 and gp120. A large percentage of CD4-gp120 contacts revolved around a cavity in gp120 in which the PHE43 residue of CD4 caps. We were able to design and synthesize a progression of small molecule compounds targeting the PHE43CD4 cavity in gp120 based on a previous CD4-mimetic, NBD-556. By either soaking or co-crystallizing the newly designed compounds bound to gp120, we were able to solve four x-ray crystal structures in order to observe the interactions with the binding cavity on the atomic level. Using x-ray crystal structure, isothermal calorimetry and viral binding assay to guide design, we were able to improve the binding affinity more than 30 fold compared to the original NBD-556. Our most potent compound DMJ-II-121-R,R is able to bind to gp120 at a Kd of 0.11 micromolar and specifically block HIV-1 entry at an IC50 of 2.3 micromolar. Along with improved potency, the new design alleviated the agonistic properties of the original NBD-556, which was inducing gp120 to bind to the coreceptors on the host cell instead of blocking the progression of the HIV lifecycle. In parallel, we also utilized the soakable gp120 crystal system to screen a library of 352 fragments of various shapes using x-ray crystallography to detect and identify two positive hits, benzimidazole and 3-hydroxyphenylacetic acid. The possible leads from the two identified fragments along with our improved potency of NBD-556 based derivatives offer valuable insight to guide us on the development toward a subnanomolar small molecular antagonist of gp120-CD4 binding

In Silico Identification of Structure Requirement for Novel Thiazole and Oxazole Derivatives as Potent Fructose 1,6-Bisphosphatase Inhibitors

Fructose 1,6-bisphosphatase (FBPase) has been identified as a drug discovery target for lowering glucose in type 2 diabetes mellitus. In this study, a large series of 105 FBPase inhibitors were studied using a combinational method by 3D-QSAR, molecular docking and molecular dynamics simulations for a further improvement in potency. The optimal 3D models exhibit high statistical significance of the results, especially for the CoMFA results with rncv2, q2 values of 0.986, 0.514 for internal validation, and rpred2, rm2 statistics of 0.902, 0.828 statistics for external validation. Graphic representation of the results, as contoured 3D coefficient plots, also provides a clue to the reasonable modification of molecules. (1) Substituents with a proper length and size at the C5 position of the thiazole core are required to enhance the potency; (2) A small and electron-withdrawing group at the C2 position linked to the thiazole core is likely to help increase the FBPase inhibition; (3) Substituent groups as hydrogen bond acceptors at the C2 position of the furan ring are favored. In addition, the agreement between 3D-QSAR, molecular docking and molecular dynamics simulation proves the rationality of the developed models. These results, we hope, may be helpful in designing novel and potential FBPase inhibitors

Evaluation of Cytotoxic Effects and Underlying Mechanism of Phenolic Compounds on Breast Cancer Cell Lines

Breast cancer (BC) is the second most common cancer that causes higher mortality rates worldwide. It is a complex and heterogeneous disease with a median survival range of around three years. Breast cancer patients' overall survival has increased due to using chemotherapeutic medicines, namely anthracyclines and taxanes. However, drug resistance and subsequent progression of this disease were still observed in metastatic patients. Furthermore, the efficacy failure of even today's sophisticated chemotherapeutics negatively impacts breast cancer patients with side effects, highlighting the urgent need for the development of nontoxic medications, that have low side effects, and are patient-friendly. Tumor cell death has been associated with the activation of apoptotic signal transduction pathways in cancer cells, such as the intrinsic and/or extrinsic pathways. Thus, understanding the molecular mechanism of apoptosis opens the future perspective for drug development for breast cancer treatment. The present study focuses on the possibility of using newly synthesized indoline analogs as targeted therapy for breast cancer, which could selectively induce apoptosis in cancer cells. Advances in anticancer drug discovery using broad-spectrum drugs, such as substituted alkylamino phenolic rings or indoline rings, have emerged as promising molecules. Thus, investigating the effects of these compounds in inducing apoptosis would provide opportunities that directly evade the significant challenges in current breast cancer therapies. The present research work focuses on the in-vitro analysis of the anti-breast cancer activity of three novel indoline derivatives, 2-((1, 2, 3, 4-Tetrahydroquinolin-1-yl) (4 methoxyphenyl) methyl) phenol (THMPP), 2-((3,4-Dihydroquinolin-1(2H)-yl) (ptolyl)methyl)phenol)(THTMP) and N-(2-hydroxy-5-nitrophenyl (4’-methylphenyl)methyl) indoline (HNPMI). The present study’s findings have been published in four publications, each of which highlights the mechanism of action of each compound’s cytotoxic potential, its apoptotic induction potential, the regulation of the genes involved in the Epithelial Growth Factor Receptor (EGFR) signaling pathway, and the in-silico analysis to identify the compound’s interaction with the target receptor, EGFR. Absorption, distribution, metabolism, excretion, and toxicity (ADME/ T) analysis also confirms the drug likeliness of the compound to be used as a potent anticancer drug. The compounds' cytotoxicity was tested in breast cancer cells like MCF7 (ER+PR+/HER-), SkBr3 (ER-PR-/HER+), MDA MB-231, and non-tumorous cells like HEK293 and H9C2. The first compound we analyzed was 2-((1, 2, 3, 4-Tetrahydroquinolin-1-yl) (4 methoxyphenyl) methyl) phenol (THMPP). In human breast cancer cell lines MCF- 7 and SkBr3 and non-cancerous mouse myoblast cells, H9C2, it was evaluated for its potential cytotoxicity and method of action. THMPP induced cell death in MCF- 7 and SkBr3 cells at their IC50 concentration of 83.23 μM and 113.94 μM, respectively. The toxicity was 36.4% in MCF-7 cells and 18.86% in SkBr3 cells at 10μM concentrations. Interestingly, THMPP showed the lowest percentage of cytotoxicity to H9C2 cells (0.91%) than the other breast cancer cell line. The compound induced apoptosis through increased caspase three and caspase 9 with the fold level of 0.17-fold and 0.47-fold in MCF-7 cells and 0.07 and 0.25 in SkBr3 cells, respectively. THMPP also enhanced apoptosis of the breast cancer cells, causing inter-nucleosomal DNA fragmentation, thus leading to DNA strand breakage and cell death. FACS analysis has proved that THMPP improves breast cancer cells to enter various stages of apoptosis, especially in the late apoptotic stage. With a score of 5.79 kJ/mol, molecular docking validates THMPP's substantial interaction with EGFR, predicted to activate the downstream signaling pathway. The downregulation of PI3K and S6K1 genes involved in the Phosphatidylinositol 3-Kinase (PI3K)/AKT signaling pathway, which were considerably overexpressed in cancer cells, was validated by gene expression analysis. Quantitative Structure-Activity Relationship (QSAR) analysis confirmed the toxicity of THMPP against breast cancer cells. ADME/T analysis predicts the drug-likeliness of THMPP. The second important compound that was analyzed for its anti-breast cancer activity was 2-((3,4-Dihydroquinolin-1(2H)-yl) (p-tolyl) methyl) phenol) (THTMP), the derivative of THMPP. A methyl group replaced the 4-OMe substituent of the aryl ring in THMPP to synthesize THMPP. The compound exhibits a cytotoxic effect against MCF7 and SK-BR3 cells, with IC50 values of 87.92 μM and 172.51 μM, respectively. THTMP caused cell death in breast cancer cells by regulating critical apoptosis enzymes, caspase-3 and-9, with 33 percent of cells in the late apoptotic stage after 24 hours of treatment. The significant interaction of THTMP with EGFR inhibits PI3K/S6K1 gene expression, thus enhancing the apoptotic response of the breast cancer cells. Structural validation of QSAR also confirms the anticancer property of THTMP. ADME/T screening suggested the compound’s oral availability and better intestinal absorption with acceptable metabolism and toxicity parameters. Furthermore, seven N-substituted indoline derivatives have been assessed for their ability to interact with the EGFR protein. Among the seven compounds analyzed by molecular docking, it was confirmed that the N-(2-hydroxy-5-nitrophenyl (4’- methyl phenyl) methyl) indoline (HNPMI) possesses a stronger affinity with EGFR active sites. As a result, the EGFR signaling pathway was activated, which reduced the expression of PI3K and S6K1 to about 0.4-fold and 0.3-fold, respectively, thereby inducing cell death via inter-nucleosomal DNA fragmentation. The IC50 value of HNPMI was found to be 64.10 μM in MCF-7 cells and 119.99 μM in SkBr3 cells. Furthermore, HNPMI stimulated DNA fragmentation, which was validated by FACS analysis, resulting in caspase-mediated apoptosis. Structural elucidation also revealed the bi-molecular interaction of HNPMI-EGFR, relating its activity to the anti-proliferative and apoptotic activity. Finally, a combined computational analysis was performed to predict the compounds’ interaction with the tyrosine kinase receptor of EGFR. The study revealed that the HNPMI, THMPP, and THTMP interact with the active site region of the EGFR structure (PDB ID: 1M17). The interactions include hydrogen bonds, hydrophobic interactions, pi stacking, and salt bridges. HNPMI was found to have the lowest Glide docking score among the three compounds, reflecting that it can be a better inhibitor than the other two compounds. The investigation of the MMGB/ SA and QM/MM analysis also showed a coherent pattern. It was also found that the protein-ligand complex was stable when it was simulated for 100ns. The Molecular dynamics results also revealed that the ligand interacted with the protein for more than 30% of the simulation time. The compounds also possessed good pharmacokinetic properties, which were predicted by ADME/T analysis. Overall, the study demonstrates the effect of cytotoxicity and apoptotic induction of indoline derivatives THMPP, THTMP, and HNPMI. Furthermore, our results revealed the anti-breast cancer property of all three phenolic compounds with HNPMI as the lead molecule. HNPMI was also observed to be a potent EGFR pathway inhibitor, inhibiting the PI3K/ S6K1 signaling pathway and causing cell death in breast cancer cells. Thus, HNPMI can be subjected to further clinical testing and developed as a promising therapeutic medication for the treatment of breast cancer

Elucidating Proteasome Catalytic Subunit Composition and Its Role in Proteasome Inhibitor Resistance

Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached. Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell types, less is known regarding their contribution to cellular responses to proteasome inhibitors. Importantly, previous studies in murine models have shown that individual proteasome subtypes differ in sensitivity to specific proteasome inhibitors. Furthermore, research efforts in our laboratory and others have revealed that proteasome catalytic subunit expression levels and activity profiles are altered when human cancer cells acquire resistance to proteasome inhibitors. We therefore hypothesized that changes in the relative abundances of individual proteasome subtypes contribute to the acquired resistance of cancer cells to bortezomib and carfilzomib. A major obstacle in testing our hypothesis was a lack of chemical probes suitable for use in identifying distinct proteasome subtypes. We addressed this by developing a series of bifunctional proteasome probes capable of crosslinking specific pairs of catalytic subunits colocalized within individual proteasome complexes and compatible with immunoblotting-based detection of the crosslinked subunit pairs. We confirmed the utility of these probes in discerning the identities of individual proteasome subtypes in a multiple myeloma cell line that abundantly expresses catalytic subunits of both the constitutive proteasome and immunoproteasome. Our findings indicate that constitutive proteasomes, immunoproteasomes, and intermediate proteasomes co-exist within these cells and support conclusions drawn from previous studies in other cell types. We also established non-small cell lung cancer cell line models of acquired bortezomib and carfilzomib resistance in which to test our hypothesis. Using immunoblotting and proteasome activity assays, we discovered that changes in the expression levels and activities of individual catalytic proteasome subunits were associated with the emergence of acquired resistance to bortezomib or carfilzomib. These changes were inhibitor-dependent and persisted after the selective pressure of the inhibitor was removed. Finally, results obtained using our bifunctional proteasome probes suggest that the altered abundance of an intermediate proteasome subtype is associated with acquired proteasome inhibitor resistance. Collectively, our results provide evidence linking changes proteasome composition with acquired proteasome inhibitor resistance and support the hypothesis that such changes are involved in resistance mechanisms to these inhibitors

PBK/TOPK INHIBITOR OTS964 RESISTANCE IS MEDIATED BY ABCG2- AND ABCB1-DEPENDENT TRANSPORT FUNCTION IN CANCER

Accumulating evidence has suggested that multi-drug resistance (MDR) in cancer cells is a phenotype whereby cancer cells have attenuated sensitivity to drugs. ATP-binding cassette super-family G member 2 (ABCG2/BCRP) and ATP-binding cassette sub-family B member 1 (ABCB1/P-gp) are members of the ATP-binding cassette (ABC) transporter family and involved in MDR. OTS964 is a potent inhibitor targeting to PDZ-binding kinase (PBK)/T-lymphokine-activated killer cell-originated protein kinase (TOPK). Herein, we aimed to explore the relationship between MDR-associated ABC transporters, including ABCG2 and ABCB1, and the regulation of OTS964 efficacy. PBK/TOPK inhibitor OTS964 resistance is mediated by ABCG2-dependent transport function in cancer: in vitro study The efficacy of OTS964 is limited in drug-selected and drug resistant gene-transfected cells, which overexpress ABCG2, compared to those of corresponding drug-sensitive cells. Also, a verified ABCG2 inhibitor Ko143 can re-sensitize the acquired resistance to OTS964. In mechanism-based studies, OTS964 shows inhibitory effect on the efflux function mediated by ABCG2. Furthermore, OTS964 stimulates ATPase activity of ABCG2 and upregulates ABCG2 expression, resulting in enhanced resistance to substrate-drugs transported by ABCG2. The in silico molecular docking analysis suggested that OTS964 interacts with drug-binding pocket of ABCG2. PBK/TOPK inhibitor OTS964 resistance is mediated by ABCB1-dependent transport function in cancer: in vitro and in vivo study The overexpression of ABCB1 significantly desensitizes both drug-selected and gene-transfected cell lines, which overexpress ABCB1, to OTS964 and that this drug resistance can be antagonized by a verified ABCB1 inhibitor verapamil. Also, a similar trend was observed in tumor-bearing mouse model. In mechanistic studies, OTS964 inhibits the efflux function mediated by ABCB1. Moreover, OTS964 stimulates ATPase activity and expression levels of ABCB1, leading to induced resistance to substrate-drugs transported by ABCB1. OTS964 receives a comparable affinity score and can dock into the substrate-binding site of human ABCB1 protein. Altogether, OTS964 is susceptible to ABCG2- and ABCB1-mediated drug resistance, and that this effect can be antagonized by known inhibitors. Our findings strongly support the importance of monitoring the level of ABCG2 and ABCB1 in cancer patients under OTS964 treatment. These findings may also serve as a valuable indication for follow-up clinical investigation on potential use of OTS964

Drug Repurposing

This book focuses on various aspects and applications of drug repurposing, the understanding of which is important for treating diseases. Due to the high costs and time associated with the new drug discovery process, the inclination toward drug repurposing is increasing for common as well as rare diseases. A major focus of this book is understanding the role of drug repurposing to develop drugs for infectious diseases, including antivirals, antibacterial and anticancer drugs, as well as immunotherapeutics

Inhibitors of Human ABCG2: From Technical Background to Recent Updates With Clinical Implications

The ATP-binding cassette transporter G2 (ABCG2; also known as breast cancer resistance protein, BCRP) has been suggested to be involved in clinical multidrug resistance (MDR) in cancer like other ABC transporters such as ABCB1 (P-glycoprotein). As an efflux pump exhibiting a broad substrate specificity localized on cellular plasma membrane, ABCG2 excretes a variety of endogenous and exogenous substrates including chemotherapeutic agents, such as mitoxantrone and several tyrosine kinase inhibitors. Moreover, in the normal tissues, ABCG2 is expressed on the apical membranes and plays a pivotal role in tissue protection against various xenobiotics. For this reason, ABCG2 is recognized to be an important determinant of the pharmacokinetic characteristics of its substrate drugs. Although the clinical relevance of reversing the ABCG2-mediated MDR has been inconclusive, an appropriate modulation of ABCG2 function during chemotherapy should logically enhance the efficacy of anti-cancer agents by overcoming the MDR phenotype and/or improving their pharmacokinetics. To confirm this possibility, considerable efforts have been devoted to developing ABCG2 inhibitors, although there is no clinically available substance for this purpose. As a clue for addressing this issue, this mini-review provides integrated information covering the technical backgrounds necessary to evaluate the ABCG2 inhibitory effects on the target compounds and a current update on the ABCG2 inhibitors. This essentially includes our recent findings, as we serendipitously identified febuxostat, a well-used agent for hyperuricemia as a strong ABCG2 inhibitor, that possesses some promising potentials. We hope that an overview described here will add value to further studies involving in the multidrug transporters