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.

MEIS inhibitors reduce the viability of primary leukemia cells and Stem cells by inducing apoptosis.

Leukemia stem cells (LSCs) exhibit self-renewal, resistance to standard treatments, and involvement in leukemia relapse. Higher Myeloid Ecotropic Integration Site-1 (MEIS1) expression in leukemic blast samples has been linked to resistance to conventional treatment. We studied the MEIS1 and associated factors in relapsed LSCs and assessed the effect of recently developed MEIS inhibitors (MEISi). Meis1 gene expression was found to be higher in patients with leukemia and relapsed samples. The majority of CD123+ and CD34+ LSCs demonstrated higher MEIS1/2/3 content. Depending on the patient chemotherapy regimen, Meis1 expression increased in relapsed samples. Although there are increased Meis2, Meis3, Hoxa9, Pbx1, or CD34 expressions in the relapsed patients, they are not correlated with Meis1 content in every patient or regimen. MEISi has reduced MEIS1 transcriptional activity and LSC cell survival by apoptosis. Pharmacological targeting with MEISi in LSCs could have a potential effect in limiting leukemia relapse and chemotherapeutic resistance.

Development of novel 1,2,4-triazole containing compounds with anticancer and potent anti-CB1 activity.

There is still an unmet need for novel and improved anti-cancer compounds. Nitrogen atoms have heterocyclic ring moieties, which have been shown to have powerful anticancer properties in both natural and synthetic derivatives. Due to their dipole character, hydrogen bonding capacity, rigidity and solubility, 1,2,4-triazoles are particularly effective pharmacophores, interacting with biological receptors with high affinity. Thus, novel 1,2,4-triazole-containing molecular derivatives were synthesized using green chemistry methods, microwave irradiation and ultrasonication, and these methods' operational simplicity and maximum greener synthetic efficiency with green chemistry metrics calculations will be attractive for academic and industrial research and tested against three distinct human cancer cell lines including PANC1 (pancreatic cancer), DU145 (prostate cancer), MCF7 (breast cancer) and one fibroblast cell line (HDF). Here, we showed that compounds 5e and 5f were similar to CB1 antagonists in structure, binding affinity and poses. In addition, compounds 5e-g decreased the viability of pancreatic and prostate cancer cells, albeit with cytotoxicity to HDF cells. The IC50 values for PANC1 cells were between 5.9 and 7.3 µM for compounds 5e-g. Cell cycle analysis showed that the effect of compounds 5e-g in cancer cell growth was largely due to cell cycle arrest at S-phase. In sum, novel 1,2,4-triazole-containing compounds with anticancer and potent anti-CB1 activity have been developed.Communicated by Ramaswamy H. Sarma.

Newly developed MEIS inhibitor selectively blocks MEISHigh prostate cancer growth and induces apoptosis.

Prostate cancer (PCa) is the second most diagnosed cancer in males. Understanding the molecular mechanism and investigation of novel ways to block PCa growth or metastasis are vital and a medical necessity. In this study, we examined differential expression of MEIS1/2/3 and its associated factors in PCa cell lines. MEIS1/2/3 content, reactive oxygen species, and cell cycle status were analyzed in PCa cells post MEIS inhibitor (MEISi) treatments, which is developed in our laboratory as a first-in-class small molecule inhibitor. A correlation was detected between MEIS content and MEISi IC50 values of PCa cells. MEISi decreased the viability of PC-3, DU145, 22Rv-1 and LNCaP cells, and significantly increased apoptosis in parallel with the increased cellular ROS content. The efficacy of MEISi was shown to positively correlate with the levels of MEIS1/2/3 proteins and the long term exposure to MEISi elevated MEIS1/2/3 protein content in PCa cells. Our findings suggest that MEISi could be used to target PCa with high MEIS expression in order to reduce PCa viability and growth; however, more research is needed before this can be translated into clinical settings.

Therapeutic targeting and HSC proliferation by small molecules and biologicals.

Hematopoietic stem cells (HSCs) have considerably therapeutic value on autologous and allogeneic transplantation for many malignant/non-malignant hematological diseases, especially with improvement of gene therapy. However, acquirement of limited cell dose from HSC sources is the main handicap for successful transplantation. Therefore, many strategies based on the utilization of various cytokines, interaction of stromal cells, modulation of several extrinsic and intrinsic factors have been developed to promote ex vivo functional HSC expansion with high reconstitution ability until today. Besides all these strategies, small molecules become prominent with their ease of use and various advantages when they are translated to the clinic. In the last two decades, several small molecule compounds have been investigated in pre-clinical studies and, some of them were evaluated in different stages of clinical trials for their safety and efficiencies. In this chapter, we will present an overview of HSC biology, function, regulation and also, pharmacological HSC modulation with small molecules from pre-clinical and clinical perspectives.

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.

Competitive inhibition of IL-2/IL-2R has a dual effect on HSC ex vivo expansion and IL-2R (CD25) content.

Interleukin-2 (IL-2) and its receptor play a pivotal role in the regulation of immune response and possess both immune-regulatory and immune-stimulatory functions. As a cytokine of lymphoid cells, the role of IL-2 has been revealed in hematopoietic stem cell (HSC) maintenance and proper hematopoiesis. Here, we investigated that small molecule Ro 26-4550 trifluoroacetate (Ro) mediated competitive inhibition of IL-2 and its receptor alpha subunit (IL-2Rα) throughout ex vivo culture. Ro treatment induced murine and human ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). Ro treated HSPCs sustained self-renewal ability and low apoptotic activity. As a competitive inhibitor of IL-2/IL-2Rα interaction, Ro small molecule induced human HSPCs to entry into cell cycle. The proliferation of bone marrow mesenchymal stem cells (MSC) and fibroblasts were also highly increased post treatment. Besides, Ro treatment enhanced IL-2Rα (CD25) expression independent of IL-2 administration in human mPB-derived HSPCs and BM-derived HSPCs. Increased IL-2Rα (CD25) expression in BM-HSPCs was associated with the increase in the CD4+CD25+ T cell population. Xenotransplantation of immunodeficient mice with ex vivo expanded human CD34+ cells after Ro treatment revealed an efficient multi-lineage reconstitution in the recipient. These findings shed light on the role of IL-2/IL-2Rα interaction in HSC expansion, in vivo and in vitro HSC self-renewal ability and repopulation capacity as well as a possible mean for the induction of CD25 expressing cells in hematopoietic compartments.

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.

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.

The current state of validated small molecules inhibiting SARS-CoV-2 nonstructural proteins.

The current COVID-19 outbreak has had a profound influence on public health and daily life. Despite all restrictions and vaccination programs, COVID-19 still can lead to fatality due to a lack of COVID-19-specific treatments. A number of studies have demonstrated the feasibility to develop therapeutics by targeting underlying components of the viral proteome. Here we reviewed recently developed and validated small molecule inhibitors of SARS-CoV-2's nonstructural proteins. We described the validation level of identified compounds specific for SARS-CoV-2 in the presence of in vitro and in vivo supporting data. The mechanisms of pharmacological activity, as well as approaches for developing improved SARS-CoV-2 NSP inhibitors have been emphasized.

Screening of the small molecule library of Meinox enables the identification of anticancer compounds in pathologically distinct cancers.

Small molecules are widely used for the modulation of the molecular basis of diseases. This makes them the perfect tool for discovering and developing new therapeutics. In this work, we have established a library of small molecules in house and characterized its molecular and druglike properties. We have shown that most small molecules have molecular weights less than 450. They have pharmaceutically relevant cLogP, cLogS, and druglikeness value distributions. In addition, Meinox's small molecule library contained small molecules with polar surface areas that are less than 60 square angstroms, suggesting their potent ability to cross the blood-brain barrier. Meinox's small molecule library was also tested in vitro for pathologically distinct forms of cancer, including pancreatic adenocarcinoma PANC1, breast carcinoma MCF7, and lymphoblastic carcinoma RS4-11 cell lines. Analysis of this library at a dose of 1 µM allowed the discovery of potent, specific or broadly active anticancer compounds against pathologically distinct cancers. This study shows that in vitro analysis of different cancers or other phenotypic assays with Meinox small molecule library may generate novel and potent bioassay-specific compounds.

Lead Optimization and Structure-Activity Relationship Studies on Myeloid Ecotropic Viral Integration Site 1 Inhibitor.

The pivotal role of the myeloid ecotropic viral integration site 1 (MEIS1) transcriptional factor was reported in cardiac regeneration and hematopoietic stem-cell (HSC) regulation with our previous findings. MEIS1 as a promising target in the context of pharmacological inhibition, we identified a potent myeloid ecotropic viral integration site (MEIS) inhibitor, MEISi-1, to induce murine and human HSC expansion ex vivo and in vivo. In this work, we performed lead optimization on MEISi-1 by synthesizing 45 novel analogues. Structure-activity relationship studies revealed the significance of a para-methoxy group on ring A and a hydrophobic moiety at the meta position of ring B. Obtained biological data were supported by inhibitor docking and molecular dynamics simulation studies. Eleven compounds were depicted as potent inhibitors demonstrating a better inhibitory profile on MEIS1 and target genes Meis1, Hif-1α, and p21. Among those, 4h, 4f, and 4b were the most potent inhibitors. The predicted pharmacokinetics properties fulfill drug-likeness criteria. In addition, compounds exerted neither cytotoxicity on human dermal fibroblasts nor mutagenicity.

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.

SC1 limits tube formation, branching, migration, expansion and induce apoptosis of endothelial cells.

Endothelial cells (ECs) are essential in the growth and progression of the tumor cells by supplying nutrition and angiogenesis factors. Targeting ECs emerged as a major strategy to prevent the growth of tumors. Studies suggest that ERK1/2 signaling is important for endothelial cells, which could be specifically targeted by small molecule SC1. We aimed to study the effects of SC1 treatments on endothelial cell proliferation, angiogenesis, and death. To this end, we performed viability, apoptosis, cell cycle, gene expression, wound closure, tube formation, and western blot analysis in endothelial cells post SC1 treatments. Intriguingly, we found that SC1 has an antiangiogenic effect on endothelial cells, which limits the endothelial cell expansion, tube formation, branching, and migration. The proliferation is especially limited in dose dependent manner by SC1. In addition, we found that SC1 elevates the apoptosis of endothelial cells and associated pathways including BAK1, Stat1, Sox4, and Caspase1. We believe that these findings could contribute to the development of improved therapies based on the SC1 as an attractive candidate for anticancer clinical studies targeted to tumor angiogenesis.

Modulating Cas9 activity for precision gene editing.

The CRISPR/Cas9 is a RNA-guided nuclease complex that can be specifically programmed to target a user-specified DNA sequence. It has been a powerful and effective tool of genome editing. However, off-target activity of the Cas9 nuclease limits its potential use in the correction of inherited diseases and bona fide gene editing. Various protein engineering and guide RNA selection strategies have been utilized to improve Cas9-based genome-editing specificity and efficiency. We, however, have not yet achieved a degree of safety such that Cas9 gene editing approaches could be applicable in clinical settings. Here, we discuss the recently developed and precise gene editing technologies based on spCas9. Furthermore, we describe Cas9 modulating tools to increase the fidelity of the CRISPR/Cas9 system. These studies suggest that there is still a need for pharmaceutical modulation of Cas9 activity during gene editing procedures. Pharmaceutical modulation of Cas9 nuclease activity at on-target or off-target genomic loci could 1 day allow researchers to develop robust and precise therapeutical strategies in gene editing.

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.

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.