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.

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.

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.

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.

Peripheral priming induces plastic transcriptomic and proteomic responses in circulating neutrophils required for pathogen containment.

Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow are shaped within the circulation remains vague. Experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile human and murine neutrophil transcriptomes and proteomes during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to dynamic shifts dominated by conserved up-regulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB signaling-dependent up-regulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by up-regulation of distinct effector molecules upon infection. These data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation.

Multiomic analyses uncover immunological signatures in acute and chronic coronary syndromes.

Acute and chronic coronary syndromes (ACS and CCS) are leading causes of mortality. Inflammation is considered a key pathogenic driver of these diseases, but the underlying immune states and their clinical implications remain poorly understood. Multiomic factor analysis (MOFA) allows unsupervised data exploration across multiple data types, identifying major axes of variation and associating these with underlying molecular processes. We hypothesized that applying MOFA to multiomic data obtained from blood might uncover hidden sources of variance and provide pathophysiological insights linked to clinical needs. Here we compile a longitudinal multiomic dataset of the systemic immune landscape in both ACS and CCS (n = 62 patients in total, n = 15 women and n = 47 men) and validate this in an external cohort (n = 55 patients in total, n = 11 women and n = 44 men). MOFA reveals multicellular immune signatures characterized by distinct monocyte, natural killer and T cell substates and immune-communication pathways that explain a large proportion of inter-patient variance. We also identify specific factors that reflect disease state or associate with treatment outcome in ACS as measured using left ventricular ejection fraction. Hence, this study provides proof-of-concept evidence for the ability of MOFA to uncover multicellular immune programs in cardiovascular disease, opening new directions for mechanistic, biomarker and therapeutic studies.

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.