Structure Activity Relationship Studies around DB18, a Potent and Selective Inhibitor of CLK Kinases.

Three series of our lead CLK1 inhibitor DB18 have been designed, synthetized and tested against CLKs and DYRK1A kinases. Their cytotoxicity was subsequently measured on seven representative cancer cell lines. Guided by docking experiments, we focused on the less constrained part of the scaffold, and showed that drastically different substituents can be tolerated here. This work ended with the discovery of another promising derivative 12g, with IC50 = 0.004 µM in the inhibition of HsCLK1 and IC50 = 3.94 µM for the inhibition of HsDYRK1A. The SAR results are discussed in the light of extensive molecular modeling analyses. Finally, a kinome scan (463 human kinases) confirmed the outstanding selectivity of our lead compound DB18, suggesting that this scaffold is of prominent interest for selective CLK inhibitors. Altogether, these results pave the way for the development of inhibitors with novel selectivities in this family of kinases.

Synthesis and biological studies of new piperidino-1,2,3-triazole hybrids with 3-aryl isoxazole side chains.

A small library of new piperidine-triazole hybrids with 3-aryl isoxazole side chains has been designed and synthesized. Their cytotoxicity against a panel of seven cancer cell lines has been established. For the most promising compound, an IC50 value of 3.8 µM on PUMA/Bcl-xL interaction in live cancer cells was established through BRET analysis. A rationale was proposed for these results through complete molecular modelling studies.

Discovery of DB18, a potent inhibitor of CLK kinases with a high selectivity against DYRK1A kinase.

We describe in this paper the synthesis of a novel series of anilino-2-quinazoline derivatives. These compounds have been screened against a panel of eight mammalian kinases and in parallel they were tested for cytotoxicity on a representative panel of seven cancer cell lines. One of them (DB18) has been found to be a very potent inhibitor of human "CDC2-like kinases" CLK1, CLK2 and CLK4, with IC50 values in the 10-30 nM range. Interestingly, this molecule is inactive at 100 µM on the closely related "dual-specificity tyrosine-regulated kinase 1A" (DYRK1A). Extensive molecular simulation studies have been performed on the relevant kinases to explain the strong affinity of this molecule on CLKs, as well as its selectivity against DYRK1A.

Afatinib or Bevacizumab in combination with Osimertinib efficiently control tumor development in orthotopic murine models of non-small lung cancer.

Lung cancer is one of the most common and deadliest cancers. Preclinical models are essential to study new therapies and combinations taking tumor genetics into account. We have established cell lines expressing the luciferase gene from lines with varied genetic backgrounds, commonly encountered in patients with pulmonary adenocarcinoma. We have characterized these lines by testing their response to multiple drugs. Thus, we have developed orthotopic preclinical mouse models of NSCLC with very high engraftment efficiency. These models allow the easy monitoring of tumor growth, particularly in response to treatment, and of tumor cells dissemination in the body. We show that concomitant treatment with osimertinib (3rd generation tyrosine kinase inhibitor targeting mutated EGFR) and bevacizumab (anti-angiogenic targeting VEGF) can have a beneficial therapeutic effect on EGFR-mutated tumors. We also show that the addition of afatinib to osimertinib-treated tumors in escape leads to tumor growth inhibition. No such effect is observed with selumetinib or simvastatin. These preclinical mouse models therefore make it possible to test innovative therapeutic combinations and are also a tool of choice for studying resistance mechanisms.

In vitro effects of Choukroun's platelet-rich fibrin conditioned medium on 3 different cell lines implicated in dental implantology.

OBJECTIVE: The aim of this work was to determine the relevance of Choukroun's platelet-rich fibrin (PRF) in dental implantology by determining the in vitro effects of soluble factors released by PRF clot. We used 3 different cell lines implicated in dental implantology: osteoblast, keratinocyte, and fibroblast. METHODS: Cellular viability, cell proliferation, and gene expression were analyzed using PRF conditioned medium. Three different cells lines were used: SaOS2 (osteoblast), MRC5 (fibroblast), and KB (epithelial cell). RESULTS: The sulforhodamine B assay showed a significant increase in cell number for the undiluted and 1:3 diluted conditioned medium after 24 and 48 hours. There was no effect for the 1:9 dilution. Cell cycle analysis by flow cytometry confirmed the viability test results. After 48 hours, PRF conditioned medium induced gene expression in osteoblasts. Expression of osteopontin and osteocalcin, late osteogenic markers, was observed using reverse transcriptase-polymerase chain reaction (RT-PCR). CONCLUSIONS: This study establishes a model to evaluate, in vitro, the effects of soluble growth factors released by PRF clot. Our work confirmed PRF is useful in stimulating tissue healing and bone regeneration. This work should recommend Choukroun's PRF in numerous implantology clinical applications.