Development, synthesis and validation of improved c-Myc/Max inhibitors.

The pathophysiological foundations of various diseases are often subject to alteration through the utilization of small compounds, rendering them invaluable tools for the exploration and advancement of novel therapeutic strategies. Within the scope of this study, we meticulously curated a diverse library of novel small compounds meticulously designed to specifically target the c-Myc/Max complex. We conducted in vitro examinations of novel c-Myc inhibitors across a spectrum of cancer cell lines, including PANC1 (pancreatic adenocarcinoma), MCF7 (breast carcinoma), DU-145 (prostate carcinoma), and A549 (lung cancer). The initial analysis involved a 25 µM dose, which enabled the identification of potent anticancer compounds effective against a variety of tumour types. We identified c-Myc inhibitors with remarkable potency, featuring IC50 values as low as 1.6 µM and up to 40 times more effective than the reference molecule in diminishing cancer cell viability. Notably, c-Myc-i7 exhibited exceptional selectivity, displaying 37-fold and 59-fold preference for targeting prostate and breast cancers, respectively, over healthy cells. Additionally, we constructed drug-likeness models. This study underscores the potential for in vitro investigations of various tumour types using novel c-Myc inhibitors to yield ground-breaking and efficacious anticancer compounds.

BML-260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability.

Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5-benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-l-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

Development of a novel and synthetic HematoMiR technology that broadly modulates quiescence of stem cells and enhances HSC expansion.

Hematopoietic stem cell (HSCs) transplantation is the primary therapeutic modality used to treat hematopoietic disorders. It centers on the capability of a small quantity of HSCs to repopulate whole blood lineages. Along with limited availability of suitable donors, the need for sufficient number of donor HSCs is still challenging in clinical relevance. This has been addressed by ex vivo HSC expansion albeit with partial success, and thus development of an alternative strategy that could improve HSC expansion is required. To that end, we aimed to build HematoMiR, an oligo-based technology that broadly targets HSC quiescence factors. Here, we show that HematoMiRs and their combinations targeting over 50 factors involved in HSC quiescence could induce robust ex vivo murine and human HSC expansion. In particular, HematoMiR-5 treatment enhanced cell cycle through down-regulation of negative cell cycle regulators in HSCs. HematoMiR-5 treated HSPCs had reduced DNA damage during the course of ex vivo expansion. Moreover, HematoMiR-5 treatment led to sustained HSC self-renewal ability and a low apoptosis rate. In addition, HematoMiR-5 expanded HSCs demonstrated successful engraftment and repopulation capacity in the recipient animals. Furthermore, combinatorial treatments of HematoMiR-2 and 5 allowed vigorous ex vivo HSC expansion. These findings demonstrate that novel and synthetic HematoMiR technology is feasible for HSC ex vivo expansion through the sequence-dependent modulation of numerous HSC quiescence modulators.

Identification of first-in-class plasmodium OTU inhibitors with potent anti-malarial activity.

OTU proteases antagonize the cellular defense in the host cells and involve in pathogenesis. Intriguingly, P. falciparum, P. vivax, and P. yoelii have an uncharacterized and highly conserved viral OTU-like proteins. However, their structure, function or inhibitors have not been previously reported. To this end, we have performed structural modeling, small molecule screening, deconjugation assays to characterize and develop first-in-class inhibitors of P. falciparum, P. vivax, and P. yoelii OTU-like proteins. These Plasmodium OTU-like proteins have highly conserved residues in the catalytic and inhibition pockets similar to viral OTU proteins. Plasmodium OTU proteins demonstrated Ubiquitin and ISG15 deconjugation activities as evident by intracellular ubiquitinated protein content analyzed by western blot and flow cytometry. We screened a library of small molecules to determine plasmodium OTU inhibitors with potent anti-malarial activity. Enrichment and correlation studies identified structurally similar molecules. We have identified two small molecules that inhibit P. falciparum, P. vivax, and P. yoelii OTU proteins (IC50 values as low as 30 nM) with potent anti-malarial activity (IC50 of 4.1-6.5 µM). We also established enzyme kinetics, druglikeness, ADME, and QSAR model. MD simulations allowed us to resolve how inhibitors interacted with plasmodium OTU proteins. These findings suggest that targeting malarial OTU-like proteases is a plausible strategy to develop new anti-malarial therapies.

The Historical Relationship Between Meis1 and Leukemia.

Acute leukemia (AL) is a poor progressive resistant hematological disease, which has different subtypes and immunophenotypic properties according to leukemic blasts. AL is caused by genetic changes and associated with leukemia stem cells (LSCs), which determine its prognosis and endurance. LSCs are thought to be hematopoietic progenitor and stem cell (HPSCs)-like cells that underwent a malignant transformation. In addition to their low number, LSCs have the characteristics of self-renewal, resistance to chemotherapy, and relapse of leukemia. The myeloid ecotropic integration site-1 (MEIS1) protein is a member of the three-amino acid loop extension (TALE) family of homeodomain (HD) proteins that can bind to DNA sequence-specific manner. Studies have shown that overexpression of MEIS1 and associated cofactors involves tumorigenesis of numerous cancers. Historically, increased expression of Meis1 transcript as well as protein has been determined in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients. Moreover, resistance to conventional chemotherapy was observed in leukemic blast samples with high Meis1 content. In this review article, the molecular mechanism of the oncological role of the MEIS1 protein in leukemia and LSC is discussed. In addition, it was suggested that MEIS1 protein could be utilized as a possible treatment target in leukemia with an emphasis on the inhibition of MEIS1, which is overexpressed in LSC.

Identification of Small Molecules That Enhance the Expansion of Mesenchymal Stem Cells Originating from Bone Marrow.

Mesenchymal stem cells (MSCs) have been shown to be promising for regenerative medicines with their immunomodulatory characteristics. They may be obtained from a variety of tissue types, including umbilical cord, adipose tissue, dental tissue, and bone marrow (BM). BM-MSCs are challenging in terms of their ex vivo expansion capability. Thus, we aimed to improve the expansion of BM-MSCs with small molecule treatments. We tested about forty small molecules that are potent quiescence modulators, and determined their efficacy by analysis of cell viability, cell cycle, and apoptosis in BM-MSCs. We also examined gene expression for selected small molecules to explore essential molecular pathways. We observed that treatment with SB203580 increased BM-MSCs expansion up to two fold when used for 5 days. SB203580 decreased the proportion of cells in the G1 phase of the cell cycle and substantially increased the ratio of cells in the S-G2-M phase. Enhanced MSC expansion with SB203580 therapy was associated with the lower expression of CDKIs like p15, p18, p19, p21, p27, and p57. In conclusion, we have developed a new approach to facilitate the expansion of BM-MSCs. These results could enhance autologous and immunomodulation therapy involving BM-MSCs.

Gene editing and RNAi approaches for COVID-19 diagnostics and therapeutics.

The novel coronavirus pneumonia (COVID-19) is a highly infectious acute respiratory disease caused by Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV-2) (Prec Clin Med 2020;3:9-13, Lancet 2020;395:497-506, N. Engl J Med 2020a;382:1199-207, Nature 2020;579:270-3). SARS-CoV-2 surveillance is essential to controlling widespread transmission. However, there are several challenges associated with the diagnostic of the COVID-19 during the current outbreak (Liu and Li (2019), Nature 2020;579:265-9, N. Engl J Med 2020;382:727-33). Firstly, the high number of cases overwhelms diagnostic test capacity and proposes the need for a rapid solution for sample processing (Science 2018;360:444-8). Secondly, SARS-CoV-2 is closely related to other important coronavirus species and subspecies, so detection assays can give false-positive results if they are not efficiently specific to SARS-CoV-2. Thirdly, patients with suspected SARS-CoV-2 infection sometimes have a different respiratory viral infection or co-infections with SARS-CoV-2 and other respiratory viruses (MedRxiv 2020a;1-18). Confirmation of the COVID-19 is performed mainly by virus isolation followed by RT-PCR and sequencing (N. Engl J Med 2020;382:727-33, MedRxiv 2020a, Turkish J Biol 2020;44:192-202). The emergence and outbreak of the novel coronavirus highlighted the urgent need for new therapeutic technologies that are fast, precise, stable, easy to manufacture, and target-specific for surveillance and treatment. Molecular biology tools that include gene-editing approaches such as CRISPR-Cas12/13-based SHERLOCK, DETECTR, CARVER and PAC-MAN, antisense oligonucleotides, antisense peptide nucleic acids, ribozymes, aptamers, and RNAi silencing approaches produced with cutting-edge scientific advances compared to conventional diagnostic or treatment methods could be vital in COVID-19 and other future outbreaks. Thus, in this review, we will discuss potent the molecular biology approaches that can revolutionize diagnostic of viral infections and therapies to fight COVID-19 in a highly specific, stable, and efficient way.

Small molecule-mediated modulation of ubiquitination and neddylation improves HSC function ex vivo.

Hematopoietic stem cells (HSCs) are particularly characterized by their quiescence and self-renewal. Cell cycle regulators tightly control quiescence and self-renewal capacity. Studies suggest that modulation of ubiquitination and neddylation could contribute to HSC function via cyclin-dependent kinase inhibitors (CDKIs). S-phase kinase-associated protein 2 (SKP2) is responsible for ubiquitin-mediated proteolysis of CDKIs. Here, we modulated overall neddylation and SKP2-associated ubiquitination in HSCs by using SKP2-C25, an SKP2 inhibitor, and MLN4924 (Pevonedistat) as an inhibitor of the NEDD8 system. Treatments of SKP2-C25 and MLN4924 increased both murine and human stem and progenitor cell (HSPC) compartments. This is associated with the improved quiescence of murine HSC by upregulation of p27 and p57 CDKIs. A colony-forming unit assay showed an enhanced in vitro self-renewal potential post inhibition of ubiquitination and neddylation. In addition, MLN4924 triggered the mobilization of bone marrow HSPCs to peripheral blood. Intriguingly, MLN4924 treatment could decrease the proliferation of murine bone marrow mesenchymal stem cells or endothelial cells. These findings shed light on the contribution of SKP2, and associated ubiquitination and neddylation in HSC maintenance, self-renewal, and expansion.

Identification of Novel and Potent Modulators Involved in Neonatal Cardiac Regeneration.

Neonatal mammalian heart has been shown to possess the capacity to regenerate substantially after an injury. This remarkable regenerative capacity is lost in a week. This transition has been marked with cardiomyocyte cell cycle arrest and induction of fibrotic response similar to what occurs after myocardial infarction in adult hearts. Recent studies outlined the function of several cardiogenic factors that play a pivotal role in neonatal cardiac regeneration. However, underlying molecular mechanisms of neonatal cardiac regeneration and other cardiogenic factors remained elusive. Here, we investigated the involvement of novel putative cardiogenic factors in neonatal cardiac regeneration and cardiomyocyte cell cycle withdrawal. We have shown that Cbl, Dnmt3a, and Itch are significantly downregulated during neonatal cardiac regeneration process after cardiac injury in vivo. Intriguingly, several of studied factors are upregulated in non-regenerative period of 7-day-old mice after cardiac injury. Knockdown of Cbl, Dnmt3a and Itch in rat neonatal cardiomyocytes lead to the induction of cardiomyocyte proliferation. Cardiomyocyte proliferation accompanies upregulation of positive regulators of cardiomyocyte division and downregulation of CDKIs. Taken together, our findings suggest that Cbl, Dnmt3a, and Itch may be involved in the regulation of cardiomyocyte cell cycle withdrawal and may represent new targets for the induction of cardiac regeneration.

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment.

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca2+ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K+ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.

CASIN and AMD3100 enhance endothelial cell proliferation, tube formation and sprouting.

Endothelial dysfunction is prominent in atherosclerosis, hypertension, diabetes, peripheral and cardiovascular diseases, and stroke. Novel therapeutic approaches to these conditions often involve development of tissue-engineered veins with ex vivo expanded endothelial cells. However, high cell number requirements limit these approaches to become applicable to clinical applications and highlight the requirement of technologies that accelerate expansion of vascular-forming cells. We have previously shown that novel small molecules could induce hematopoietic stem cell expansion ex vivo. We hypothesized that various small molecules targeting hematopoietic stem cell quiescence and mobilization could be used to induce endothelial cell expansion and angiogenesis due to common origin and shared characteristics of endothelial and hematopoietic cells. Here, we have screened thirty-five small molecules and found that CASIN and AMD3100 increase endothelial cell expansion up to two-fold and induce tube formation and ex vivo sprouting. In addition, we have studied how CASIN and AMD3100 affect cell migration, apoptosis and cell cycle of endothelial cells. CASIN and AMD3100 upregulate key endothelial marker genes and downregulate a number of cyclin dependent kinase inhibitors. These findings suggest that CASIN and AMD3100 could be further tested in the development of artificial vascular systems and vascular gene editing technologies. Furthermore, these findings may have potential to contribute to the development of alternative treatment methods for diseases that cause endothelial damage.

Developments in Artificial Platelet and Erythroid Transfusion Products.

Platelet and blood transfusions have vital importance to the lives of many patients. Platelet transfusions are a life-saving intervention by reducing risk of bleeding in thrombocytopenic patients. Due to the short shelf life of platelets and their limited availability, researchers have developed various platelet transfusion production technologies. Understanding the cellular and biophysical mechanisms of platelet release is particularly important for development of platelet transfusion products (PTPs) and to translate them to clinical applications in patients requiring platelet infusion. Similarly, due to donor dependence and increased clinical need of blood transfusions, studies on the erythroid transfusion products (ETPs) have recently gained momentum. This led to development of ETP technologies involving differentiation of stem cells to fully functional erythrocytes in vitro. During megakaryopoiesis or erythropoiesis, various stimulatory factors, growth factors, transcription factors, and biophysical conditions have been shown to play a crucial role in the formation final blood products. Thus, understanding of the in vivo mechanisms of platelet release and erythrocyte maturation is particularly important for mimicking these conditions in vitro. This review focuses on latest and up-to-date information about the innovations in PTP and ETP technologies. We also discuss some of the recent fundamental findings that have changed our understanding of in vivo platelet release and blood formation. Human bone marrow acts as a source of cells required for erythropoiesis and megakaryopoeiesis. Understanding of molecular mechanism and physiology of these vital and curitial events allowed us to mimic these conditions ex vivo and to develop artificial platelet and erythroid transfusion production technologies.

Meis1 regulates postnatal cardiomyocyte cell cycle arrest.

The neonatal mammalian heart is capable of substantial regeneration following injury through cardiomyocyte proliferation. However, this regenerative capacity is lost by postnatal day 7 and the mechanisms of cardiomyocyte cell cycle arrest remain unclear. The homeodomain transcription factor Meis1 is required for normal cardiac development but its role in cardiomyocytes is unknown. Here we identify Meis1 as a critical regulator of the cardiomyocyte cell cycle. Meis1 deletion in mouse cardiomyocytes was sufficient for extension of the postnatal proliferative window of cardiomyocytes, and for re-activation of cardiomyocyte mitosis in the adult heart with no deleterious effect on cardiac function. In contrast, overexpression of Meis1 in cardiomyocytes decreased neonatal myocyte proliferation and inhibited neonatal heart regeneration. Finally, we show that Meis1 is required for transcriptional activation of the synergistic CDK inhibitors p15, p16 and p21. These results identify Meis1 as a critical transcriptional regulator of cardiomyocyte proliferation and a potential therapeutic target for heart regeneration.

Developments in Hematopoietic Stem Cell Expansion and Gene Editing Technologies.

Hematopoietic stem cells (HSCs) are rare cells, which housed in the adult bone marrow. They maintain all types of differentiated blood cells throughout life. Due to limited availability of HSCs for transplantation, treatment of various inherited bone marrow disorders and anemia requires the development of HSC expansion and gene editing technologies. To this end, various studies addressed the use of cytokines and growth factors for HSC expansion. Major hurdle with these studies was found to be spontaneous differentiation of HSCs into different lineages during ex vivo procedure. In addition, cost efficient approaches were needed. Thus, studies move on to the identification of small molecules and development of RNA interference technologies with potential to enhance cell cycle progression and block inhibitory signaling mechanisms during ex vivo HSC expansion as well as single cell expansion of HSCs following gene editing studies. This review aims to highlight developments in hematopoietic stem cells expansion and gene editing technologies.

Stem Cells in Regenerative Cardiology.

The common prevalence of heart failure and limitations in its treatment are leading cause of attention and interest towards the induction of cardiac regeneration with novel approaches. Recent studies provide growing evidence regarding bona fide cardiac regeneration post genetic manipulations, administration of stimulatory factors and myocardial injuries in animal models and human studies. To this end, stem cells of different sources have been tested to treat heart failure for the development of cellular therapies. Endogenous and exogenous stem cells sources used in regenerative cardiology have provided a proof of concept and applicability of cellular therapies in myocardial improvement. Recent clinical studies, especially, based on the endogenous cardiac progenitor and stem cells highlighted the possibility to regenerate lost cardiomyocytes in the myocardium. This review discusses emerging concepts in cardiac stem cell therapy, their sources and route of administration, and plausibility of de novo cardiomyocyte formation.

Mitochondrial metabolism in hematopoietic stem cells requires functional FOXO3.

Hematopoietic stem cells (HSC) are primarily dormant but have the potential to become highly active on demand to reconstitute blood. This requires a swift metabolic switch from glycolysis to mitochondrial oxidative phosphorylation. Maintenance of low levels of reactive oxygen species (ROS), a by-product of mitochondrial metabolism, is also necessary for sustaining HSC dormancy. Little is known about mechanisms that integrate energy metabolism with hematopoietic stem cell homeostasis. Here, we identify the transcription factor FOXO3 as a new regulator of metabolic adaptation of HSC. ROS are elevated in Foxo3(-/-) HSC that are defective in their activity. We show that Foxo3(-/-) HSC are impaired in mitochondrial metabolism independent of ROS levels. These defects are associated with altered expression of mitochondrial/metabolic genes in Foxo3(-/-) hematopoietic stem and progenitor cells (HSPC). We further show that defects of Foxo3(-/-) HSC long-term repopulation activity are independent of ROS or mTOR signaling. Our results point to FOXO3 as a potential node that couples mitochondrial metabolism with HSC homeostasis. These findings have critical implications for mechanisms that promote malignant transformation and aging of blood stem and progenitor cells.

Profilin 1 is essential for retention and metabolism of mouse hematopoietic stem cells in bone marrow.

How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we demonstrated that the deletion of the cytoskeleton-modulating protein profilin 1 (pfn1) in hematopoietic stem cell (HSCs) led to bone marrow failure, loss of quiescence, and mobilization and apoptosis of HSCs in vivo. A switch from glycolysis to mitochondrial respiration with increased reactive oxygen species (ROS) level was also observed in HSCs on pfn1 deletion. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that the metabolism is mechanistically linked to the cell cycle quiescence of stem cells. The actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Our study provided evidence that pfn1 at least partially acts through the axis of pfn1/Gα13/EGR1 to regulate stem cell retention and metabolism in the bone marrow.

Fluorometric CCHFV OTU protease assay with potent inhibitors.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay.

Fluorometric RdRp assay with self-priming RNA.

There is an outmost need for the identification of specific antiviral compounds. Current antivirals lack specificity, making them susceptible to off-target effects, and highlighting importance of development of assays to discover antivirals targeting viral specific proteins. Previous studies for identification of inhibitors of RNA-dependent RNA polymerase (RdRp) mostly relied on radioactive methods. This study describes a fluorometric approach to assess in vitro activity of viral RdRp for drug screening. Using readily available DNA- and RNA-specific fluorophores, we determined an optimum fluorometric approach that could be used in antiviral discovery specifically for RNA viruses by targeting RdRp. Here, we show that double-stranded RNA could be successfully distinguished from single-stranded RNA. In addition, we provide a strategy based on self-priming RNA to assess RdRp activity.