The small-molecule protein ligand interface stabiliser E7820 induces differential cell line specific responses of integrin α2 expression

Abstract Background The mechanism of small-molecule stabilised protein-protein interactions is of growing interest in the pharmacological discovery process. A plethora of different substances including the aromatic sulphonamide E7820 have been identified to act by such a mechanism. The process of E7820 induced CAPERα degradation and the resultant transcriptional down regulation of integrin α2 expression has previously been described for a variety of different cell lines and been made responsible for E7820’s antiangiogenic activity. Currently the application of E7820 in the treatment of various malignancies including pancreas carcinoma and breast cancer is being investigated in pre-clinical and clinical trials. It has been shown, that integrin α2 deficiency has beneficial effects on bone homeostasis in mice. To transfer E7820 treatment to bone-related pathologies, as non-healing fractures, osteoporosis and bone cancer might therefore be beneficial. However, at present no data is available on the effect of E7820 on osseous cells or skeletal malignancies. Methods Pre-osteoblastic (MC3T3 and Saos-2) cells and endothelial (eEnd2 cells and HUVECs) cells, each of human and murine origin respectively, were investigated. Vitality assay with different concentrations of E7820 were performed. All consecutive experiments were done at a final concentration of 50 ng/ml E7820. The expression and production of integrin α2 and CAPERα were investigated by quantitative real-time PCR and western blotting. Expression of CAPERα splice forms was differentiated by semi-quantitiative reverse transcriptase PCR. Results Here we present the first data showing that E7820 can increase integrin α2 expression in the pre-osteoblast MC3T3 cell line whilst also reproducing canonical E7820 activity in HUVECs. We show that the aberrant activity of E7820 in MC3T3 cells is likely due to differential activity of CAPERα at the integrin α2 promoter, rather than due to differential CAPERα degradation or differential expression of CAPERα spliceforms. Conclusion The results presented here indicate that E7820 may not be suitable to treat certain malignancies of musculoskeletal origin, due to the increase in integrin α2 expression it may induce. Further investigation of the differential functioning of CAPERα and the integrin α2 promoter in cells of various origin would however be necessary to more clearly differentiate between cell lines that will positively respond to E7820 from those that will not.

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Faculty Opinions recommendation of Importance of Rigidity in Designing Small Molecule Drugs To Tackle Protein-Protein Interactions (PPIs) through Stabilization of Desired Conformers.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732129727.793539183 ◽

2017 ◽

Author(s):

John Lowe

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Small Molecule Drugs

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Elucidating Protein-protein Interactions Through Computational Approaches and Designing Small Molecule Inhibitors Against them for Various Diseases

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666181025114903 ◽

2018 ◽

Vol 18 (20) ◽

pp. 1719-1736 ◽

Cited By ~ 3

Author(s):

Sharanya Sarkar ◽

Khushboo Gulati ◽

Manikyaprabhu Kairamkonda ◽

Amit Mishra ◽

Krishna Mohan Poluri

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Small Molecule Inhibitors ◽

Computational Techniques ◽

Protein Protein Interactions ◽

Computational Tools ◽

Computational Approaches ◽

Protein Protein Interaction ◽

Wide Range ◽

Therapeutic Measures

Background: To carry out wide range of cellular functionalities, proteins often associate with one or more proteins in a phenomenon known as Protein-Protein Interaction (PPI). Experimental and computational approaches were applied on PPIs in order to determine the interacting partners, and also to understand how an abnormality in such interactions can become the principle cause of a disease. Objective: This review aims to elucidate the case studies where PPIs involved in various human diseases have been proven or validated with computational techniques, and also to elucidate how small molecule inhibitors of PPIs have been designed computationally to act as effective therapeutic measures against certain diseases. Results: Computational techniques to predict PPIs are emerging rapidly in the modern day. They not only help in predicting new PPIs, but also generate outputs that substantiate the experimentally determined results. Moreover, computation has aided in the designing of novel inhibitor molecules disrupting the PPIs. Some of them are already being tested in the clinical trials. Conclusion: This review delineated the classification of computational tools that are essential to investigate PPIs. Furthermore, the review shed light on how indispensable computational tools have become in the field of medicine to analyze the interaction networks and to design novel inhibitors efficiently against dreadful diseases in a shorter time span.

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Polyamines: Naturally occurring small molecule modulators of electrostatic protein–protein interactions

Journal of Inorganic Biochemistry ◽

10.1016/j.jinorgbio.2009.10.007 ◽

2010 ◽

Vol 104 (2) ◽

pp. 118-125 ◽

Cited By ~ 15

Author(s):

Anja Berwanger ◽

Susanne Eyrisch ◽

Inge Schuster ◽

Volkhard Helms ◽

Rita Bernhardt

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Naturally Occurring ◽

Small Molecule Modulators

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Targeting Disease with Small Molecule Inhibitors of Protein-Protein Interactions

Ideas in Chemistry and Molecular Sciences ◽

10.1002/9783527630516.ch9 ◽

2010 ◽

pp. 215-238

Author(s):

Fedor Forafonov ◽

Elena Miranda ◽

Ida Karin Nordgren ◽

Ali Tavassoli

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Small Molecule Inhibitors ◽

Protein Protein Interactions

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Dynamic Imaging of Small Molecule Induced Protein–Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach

Analytical Chemistry ◽

10.1021/acs.analchem.8b03476 ◽

2018 ◽

Vol 90 (24) ◽

pp. 14287-14293 ◽

Cited By ~ 7

Author(s):

Chan-I Chung ◽

Qiang Zhang ◽

Xiaokun Shu

Keyword(s):

Phase Transition ◽

Small Molecule ◽

Protein Interactions ◽

Living Cells ◽

Dynamic Imaging ◽

Protein Protein Interactions

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Faculty Opinions recommendation of A systematic method for identifying small-molecule modulators of protein-protein interactions.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1022011.254658 ◽

2004 ◽

Author(s):

Robert Kelley

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Systematic Method ◽

Small Molecule Modulators

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Preclinical Activity of the MPS1 Inhibitor S81694 in Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood-2019-125641 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2631-2631

Author(s):

Anna Kaci ◽

Emilie Adiceam ◽

Melanie Dupont ◽

Marine Garrido ◽

Jeannig Berrou ◽

...

Keyword(s):

Cell Cycle ◽

Cell Line ◽

Solid Tumors ◽

Cell Lines ◽

Small Molecule ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Jurkat Cells ◽

Cell Cycle Distribution ◽

Monopolar Spindle

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

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