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Journal of Medicinal Chemistry Mar 2013The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-L-cysteine as a...
The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-L-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (K(i)(app) < 10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921. They have good oral bioavailability and pharmacokinetics and induced complete tumor regression in nude mice explanted with lung cancer patient xenografts. Furthermore, they display fewer liabilities with CYP-metabolizing enzymes and hERG compared with ispinesib and SB-743921, which is important given the likely application of Eg5 inhibitors in combination therapies. We present the case for this preclinical series to be investigated in single and combination chemotherapies, especially targeting hematological malignancies.
Topics: Animals; Antineoplastic Agents; Benzamides; Butylamines; Cell Line, Tumor; Chromones; Cysteine; Kinesins; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Quinazolines; Structure-Activity Relationship; Transplantation, Heterologous
PubMed: 23394180
DOI: 10.1021/jm3014597 -
International Journal of Nanomedicine 2018Controlled inhibition of kinesin motor proteins is highly desired in the field of oncology. Among other interventions, there exists "targeted chemotherapeutic...
BACKGROUND
Controlled inhibition of kinesin motor proteins is highly desired in the field of oncology. Among other interventions, there exists "targeted chemotherapeutic regime/options" of selective Eg5 competitive and allosteric inhibitors, inducing cancer cell apoptosis and tumor regression with improved safety profiles.
RESEARCH QUESTION
Though promising, such studies are still under clinical trials, for the discovery of efficient and least harmful Eg5 inhibitors. The aim of this research was to bridge the computational modeling approach with drug design and therapy of cancer cells.
METHODS
A computational model, interfaced with the clinical data of "Eg5 dynamics" and "inhibitors" via special functions, is presented in this article. Comparisons are made for the drug efficacy, and the threshold values are predicted through numerical simulations.
RESULTS
Results are obtained to depict the dynamics induced by ispinesib, when used as an inhibitor of kinesin Eg5, on cancer cell lines.
Topics: Cell Line, Tumor; Humans; Models, Biological; Molecular Motor Proteins; Nanoparticles; Neoplasms; Probability; Stochastic Processes
PubMed: 30410329
DOI: 10.2147/IJN.S168780 -
Proceedings of the National Academy of... May 2018Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of...
Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that () KIF15 motility differs significantly from Eg5; () KIF15-IN-1 is a potent inhibitor of KIF15 motility; () MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and () pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.
Topics: Antineoplastic Agents; Cell Movement; Cell Proliferation; HeLa Cells; Humans; Kinesins; Microtubules; Neoplasms; Small Molecule Libraries; Spindle Apparatus
PubMed: 29703754
DOI: 10.1073/pnas.1801242115 -
Frontiers in Oncology 2022The mitotic kinesin Eg5 has emerged as a potential anti-mitotic target for the purposes of cancer chemotherapy. Whether clinical resistance to these inhibitors can arise...
The mitotic kinesin Eg5 has emerged as a potential anti-mitotic target for the purposes of cancer chemotherapy. Whether clinical resistance to these inhibitors can arise is unclear. We exploited HCT116 cancer cell line to select resistant clones to S-trityl-L-cysteine (STLC), an extensively studied Eg5 loop-L5 binding inhibitor. The STLC resistant clones differed in their resistance to other loop-L5 binding inhibitors but remained sensitive to the ATP class of competitive Eg5 specific inhibitors. Eg5 is still necessary for bipolar spindle formation in the resistant clones since the cells were sensitive to RNAi mediated depletion of Eg5. One clone expressing Eg5(T107N), a dominant point mutation in the P-loop of the ATP binding domain of the motor, appeared to be not only resistant but also dependent on the presence of STLC. Eg5(T107N) expression was associated also with resistance to the clinical relevant loop-L5 Eg5 inhibitors, Arry-520 and ispinesib. Ectopic expression of the Eg5(T107N) mutant in the absence of STLC was associated with strong non-exchangeable binding to microtubules causing them to bundle. Biochemical assays showed that in contrast to the wild type Eg5-STLC complex, the ATP binding site of the Eg5(T107N) is accessible for nucleotide exchange only when the inhibitor is present. We predict that resistance can be overcome by inhibitors that bind to other than the Eg5 loop-L5 binding site having different chemical scaffolds, and that allostery-dependent resistance to Eg5 inhibitors may also occur in cells and may have positive implications in chemotherapy since once diagnosed may be beneficial following cessation of the chemotherapeutic regimen.
PubMed: 36313676
DOI: 10.3389/fonc.2022.965455 -
Heliyon May 2021Computational calculations of 5-bromo-3-nitropyridine-2-carbonitrile (5B3N2C) on molecular structure and on energy are implemented using the 6-311++G(d,p) basis set by...
Computational calculations of 5-bromo-3-nitropyridine-2-carbonitrile (5B3N2C) on molecular structure and on energy are implemented using the 6-311++G(d,p) basis set by DFT/B3LYP method. The UV-Vis spectrum of 5B3N2C was obtained by TD-DFT with chloroform as a solvent. The analysis of molecular electrostatic potential (MEP) and frontier molecular orbital (FMO) were used to evaluate, the entire electron density and organic reactive sites of 5B3N2C. The electron-hole conversions were conjointly deliberated. Donor-acceptor interactions (NBO) analysis examines the intra-and intermolecular charge transfer, hyper conjugate interaction of the compound. The orbital molecular contributions are evaluated by density of states (DOS and PDOS). To discern the reactivity of the molecule, topology analyses were done. The biological prominence of the 5B3N2C molecule was investigated in a pertinent study of molecular docking with target protein 3CEJ exhibiting the centromere associated protein inhibitor property. Molecular Dynamics simulations were done to assess the stability of the complex. 5B3N2C physiochemical parameters were also compared to those of widely viable medications Ispinesib and Lonafarnib.
PubMed: 34095571
DOI: 10.1016/j.heliyon.2021.e07061 -
Frontiers in Immunology 2024Despite advancements, breast cancer outcomes remain stagnant, highlighting the need for precise biomarkers in precision medicine. Traditional TNM staging is insufficient...
BACKGROUND
Despite advancements, breast cancer outcomes remain stagnant, highlighting the need for precise biomarkers in precision medicine. Traditional TNM staging is insufficient for identifying patients who will respond well to treatment.
METHODS
Our study involved over 6,900 breast cancer patients from 14 datasets, including in-house clinical data and single-cell data from 8 patients (37,451 cells). We integrated 10 machine learning algorithms in 55 combinations and analyzed 100 existing breast cancer signatures. IHC assays were conducted for validation, and potential immunotherapies and chemotherapies were explored.
RESULTS
We pinpointed six stable Panoptosis-related genes from multi-center cohorts, leading to a robust Panoptosis-model. This model outperformed existing clinical and molecular features in predicting recurrence and mortality risks, with high-risk patients showing worse outcomes. IHC validation from 30 patients confirmed our findings, indicating the model's broader applicability. Additionally, the model suggested that low-risk patients benefit more from immunotherapy, while high-risk patients are sensitive to specific chemotherapies like BI-2536 and ispinesib.
CONCLUSION
The Panoptosis-model represents a major advancement in breast cancer prognosis and treatment personalization, offering significant insights for effectively managing a wide range of breast cancer patients.
Topics: Humans; Female; Breast Neoplasms; Prognosis; Breast; Immunotherapy; Precision Medicine
PubMed: 38504988
DOI: 10.3389/fimmu.2024.1359204 -
Scientific Reports Jun 2021Atypical teratoid rhabdoid tumor (ATRT) is an aggressive embryonal brain tumor among infants and young children. Two challenges exist for preclinical testing in ATRT....
Atypical teratoid rhabdoid tumor (ATRT) is an aggressive embryonal brain tumor among infants and young children. Two challenges exist for preclinical testing in ATRT. First, genetically quiet, ATRT is a difficult tumor to target molecularly. Tumor cells need to divide to propagate tumor growth-intercepting the common crossroads in cell cycle progression is a feasible strategy. KIF11 is needed for bipolar spindle formation in metaphase. We identified KIF11 as a universal target of all ATRT-molecular-subtypes. Ispinesib, a KIF11-inhibitor, effectively inhibited tumor proliferation in all seven cell lines. A second challenge-a major challenge in preclinical drug testing in-vivo among aggressive tumor models, is the narrow therapeutic window to administer drugs within the limited murine lifespan. Our most aggressive ATRT tumor model was lethal in all mice within ~ 1 month of tumor implantation. Such short-surviving mouse models are difficult to employ for preclinical drug testing due to the narrow time window to administer drugs. To overcome this time restriction, we developed a clinical staging system which allowed physically-fit mice to continue treatment, in contrast to the conventional method of fixed drug-dose-duration regimen in preclinical testing which will not be feasible in such short-surviving mouse models. We validated this approach in a second embryonal brain tumor, medulloblastoma. This is a clinically relevant, cost-efficient approach in preclinical testing for cancer and non-cancer disease phenotypes. Widely used preclinical mouse models are not the most accurate and lack the aggressive tumor spectrum found within a single tumor type. Mice bearing the most aggressive tumor spectrum progress rapidly in the limited murine life-span, resulting in a narrow therapeutic window to administer drugs, and are thus difficult to employ in preclinical testing. Our approach overcomes this challenge. We discovered ispinesib is efficacious against two embryonal brain tumor types.
Topics: Animals; Antineoplastic Agents; Drug Screening Assays, Antitumor; Mice; Neoplasms, Experimental; Rhabdoid Tumor
PubMed: 34079014
DOI: 10.1038/s41598-021-91167-6 -
Molecular Therapy : the Journal of the... Jan 2021While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen...
While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug's target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.
Topics: Binding Sites; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Drug Resistance; Endonucleases; Genetic Testing; Mutation; Protein Binding
PubMed: 33002419
DOI: 10.1016/j.ymthe.2020.09.025 -
Frontiers in Immunology 2024This study aims to identify precise biomarkers for breast cancer to improve patient outcomes, addressing the limitations of traditional staging in predicting treatment...
BACKGROUND
This study aims to identify precise biomarkers for breast cancer to improve patient outcomes, addressing the limitations of traditional staging in predicting treatment responses.
METHODS
Our analysis encompassed data from over 7,000 breast cancer patients across 14 datasets, which included in-house clinical data and single-cell data from 8 patients (totaling 43,766 cells). We utilized an integrative approach, applying 10 machine learning algorithms in 54 unique combinations to analyze 100 existing breast cancer signatures. Immunohistochemistry assays were performed for empirical validation. The study also investigated potential immunotherapies and chemotherapies.
RESULTS
Our research identified five consistent glutamine metabolic reprogramming (GMR)-related genes from multi-center cohorts, forming the foundation of a novel GMR-model. This model demonstrated superior accuracy in predicting recurrence and mortality risks compared to existing clinical and molecular features. Patients classified as high-risk by the model exhibited poorer outcomes. IHC validation in 30 patients reinforced these findings, suggesting the model's broad applicability. Intriguingly, the model indicates a differential therapeutic response: low-risk patients may benefit more from immunotherapy, whereas high-risk patients showed sensitivity to specific chemotherapies like BI-2536 and ispinesib.
CONCLUSIONS
The GMR-model marks a significant leap forward in breast cancer prognosis and the personalization of treatment strategies, offering vital insights for the effective management of diverse breast cancer patient populations.
Topics: Humans; Breast Neoplasms; Female; Machine Learning; Glutamine; Biomarkers, Tumor; Prognosis; Gene Expression Regulation, Neoplastic; Middle Aged; Transcriptome; Metabolic Reprogramming
PubMed: 38756785
DOI: 10.3389/fimmu.2024.1369289 -
PloS One 2013ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC)...
Screening compounds with a novel high-throughput ABCB1-mediated efflux assay identifies drugs with known therapeutic targets at risk for multidrug resistance interference.
ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [(125)I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.
Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line; Cyclosporine; Drug Resistance, Multiple; High-Throughput Screening Assays; Humans; Quinolines; Reproducibility of Results; Verapamil
PubMed: 23593196
DOI: 10.1371/journal.pone.0060334