"Keep on ROCKIn": Repurposed ROCK inhibitors to boost corneal endothelial regeneration.

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.

Activity of 1-aryl-4-(naphthalimidoalkyl) piperazine derivatives against Leishmania major and Leishmania mexicana.

A series of 1-aryl-4-(phthalimidoalkyl) piperazines and 1-aryl-4-(naphthalimidoalkyl) piperazines were retrieved from a proprietary library based on their high structural similarity to haloperidol, an antipsychotic with antiparasitic activity, and assessed as potential antileishmanial scaffolds. Selected compounds were tested for antileishmanial activity against promastigotes of Leishmania major and Leishmania mexicana in dose-response assays. Two of the 1-aryl-4-(naphthalimidoalkyl) piperazines (compounds 10 and 11) were active against promastigotes of both Leishmania species without being toxic to human fibroblasts. Their activity was found to correlate with the length of their alkyl chains. Further analyses showed that compound 11 was also active against intracellular amastigotes of both Leishmania species. In promastigotes of both Leishmania species, compound 11 induced collapse of the mitochondrial electrochemical potential and increased the intracellular Ca2+ concentration. Therefore, it may serve as a promising lead compound for the development of novel antiparasitic drugs.

2­Cl­IB­MECA regulates the proliferative and drug resistance pathways, and facilitates chemosensitivity in pancreatic and liver cancer cell lines.

Specific A3 adenosine receptor (A3AR) agonist, 2­chloro­N6­(3­iodobenzyl)­5'­N­methylcarboxamidoadenosine (2­Cl­IB­MECA), demonstrates anti­proliferative effects on various types of tumor. In the present study, the cytotoxicity of 2­Cl­IB­MECA was analyzed in a panel of tumor and non­tumor cell lines and its anticancer mechanisms in JoPaca­1 pancreatic and Hep­3B hepatocellular carcinoma cell lines were also investigated. Initially, decreased tumor cell proliferation, cell accumulation in the G1 phase and inhibition of DNA and RNA synthesis was found. Furthermore, western blot analysis showed decreased protein expression level of ß­catenin, patched1 (Ptch1) and glioma­associated oncogene homolog zinc finger protein 1 (Gli1), which are components of the Wnt/ß­catenin and Sonic hedgehog/Ptch/Gli transduction pathways. In concordance with these findings, the protein expression levels of cyclin D1 and c­Myc were reduced. Using a luciferase assay, it was revealed for the first time a decrease in ß­catenin transcriptional activity, as an early event following 2­Cl­IB­MECA treatment. In addition, the protein expression levels of multidrug resistance­associated protein 1 and P­glycoprotein (P­gp) were reduced and the P­gp xenobiotic efflux function was also reduced. Next, the enhancing effects of 2­Cl­IB­MECA on the cytotoxicity of conventional chemotherapy was investigated. It was found that 2­Cl­IB­MECA enhanced carboplatin and doxorubicin cytotoxic effects in the JoPaca­1 and Hep­3B cell lines, and a greater synergy was found in the highly tumorigenic JoPaca­1 cell line. This provides a novel in vitro rationale for the utilization of 2­Cl­IB­MECA in combination with chemotherapeutic agents, not only for hepatocellular carcinoma, but also for pancreatic cancer. Other currently used conventional chemotherapeutics, fluorouracil and gemcitabine, showed synergy only when combined with high doses of 2­Cl­IB­MECA. Notably, experiments with A3AR­specific antagonist, N­[9­Chloro­2­(2­furanyl)(1,2,4)­triazolo(1,5­c)quinazolin­5­yl]benzene acetamide, revealed that 2­Cl­IB­MECA had antitumor effects via both A3AR­dependent and ­independent pathways. In conclusion, the present study identified novel antitumor mechanisms of 2­Cl­IB­MECA in pancreatic and hepatocellular carcinoma in vitro that further underscores the importance of A3AR agonists in cancer therapy.

Current Adenosinergic Therapies: What Do Cancer Cells Stand to Gain and Lose?

A key objective in immuno-oncology is to reactivate the dormant immune system and increase tumour immunogenicity. Adenosine is an omnipresent purine that is formed in response to stress stimuli in order to restore physiological balance, mainly via anti-inflammatory, tissue-protective, and anti-nociceptive mechanisms. Adenosine overproduction occurs in all stages of tumorigenesis, from the initial inflammation/local tissue damage to the precancerous niche and the developed tumour, making the adenosinergic pathway an attractive but challenging therapeutic target. Many current efforts in immuno-oncology are focused on restoring immunosurveillance, largely by blocking adenosine-producing enzymes in the tumour microenvironment (TME) and adenosine receptors on immune cells either alone or combined with chemotherapy and/or immunotherapy. However, the effects of adenosinergic immunotherapy are not restricted to immune cells; other cells in the TME including cancer and stromal cells are also affected. Here we summarise recent advancements in the understanding of the tumour adenosinergic system and highlight the impact of current and prospective immunomodulatory therapies on other cell types within the TME, focusing on adenosine receptors in tumour cells. In addition, we evaluate the structure- and context-related limitations of targeting this pathway and highlight avenues that could possibly be exploited in future adenosinergic therapies.

Substituted dienes prepared from betulinic acid - Synthesis, cytotoxicity, mechanism of action, and pharmacological parameters.

A set of new substituted dienes were synthesized from betulinic acid by its oxidation to 30-oxobetulinic acid followed by the Wittig reaction. Cytotoxicity of all compounds was tested in vitro in eight cancer cell lines and two noncancer fibroblasts. Almost all dienes were more cytotoxic than betulinic acid. Compounds 4.22, 4.30, 4.33, 4.39 had IC50 below 5 µmol/L; 4.22 and 4.39 were selected for studies of the mechanism of action. Cell cycle analysis revealed an increase in the number of apoptotic cells at 5 × IC50 concentration, where activation of irreversible changes leading to cell death can be expected. Both 4.22 and 4.39 led to the accumulation of cells in the G0/G1 phase with partial inhibition of DNA/RNA synthesis at 1 × IC50 and almost complete inhibition at 5 × IC50. Interestingly, compound 4.39 at 5 × IC50 caused the accumulation of cells in the S phase. Higher concentrations of tested drugs probably inhibit more off-targets than lower concentrations. Mechanisms disrupting cellular metabolism can induce the accumulation of cells in the S phase. Both compounds 4.22 and 4.39 trigger selective apoptosis in cancer cells via intrinsic pathway, which we have demonstrated by changes in the expression of the crucial apoptosis-related protein. Pharmacological parameters of derivative 4.22 were superior to 4.39, therefore 4.22 was the finally selected candidate for the development of anticancer drug.