Fatih Kocabas

Background/aim: Hematopoietic stem cells (HSCs) are essential for treating hematological disorders; however, their low abundance and limited proliferative capacity restrict their clinical use. This study investigated whether pifithrin-α (PFT-α), a small-molecule p53 inhibitor with known genoprotective properties, could enhance the ex vivo expansion of HSCs. Materials and methods: Mouse lineage-negative (Lin⁻) cells were isolated by magnetic depletion, while human umbilical cord blood (hUCB) and mouse bone marrow (mBM) mononuclear cells were obtained using density gradient centrifugation. CD34⁺ cells were subsequently isolated by fluorescence-activated cell sorting using an antimouse CD34 antibody. The isolated cells were cultured in StemSpan SFEM II (STEMCELL Technologies Inc., Vancouver, BC, Canada) medium supplemented with mouse stem cell factor, thrombopoietin, and Flt3-L for 7 days. PFT-α was administered throughout the culture period. After 7 days, mouse cells were analyzed for Lin⁻Sca-1⁺c-Kit⁺ (LSK) and LSK CD34^low populations, whereas human HSCs were evaluated for CD34 and CD133 expression. Additional assessments included apoptosis analysis, cell cycle distribution, and in vivo engraftment in severe combined immunodeficient (SCID) mice. Results: PFT-α treatment resulted in a threefold increase in HSC populations derived from mouse and hUCB samples (p < 0.05; p < 0.01) and a twofold increase in mBM-derived HSC populations (p < 0.05). Apoptosis levels tended to decrease, and most cells remained in the G0/G1 phase, consistent with the maintenance of stem cell characteristics. Expanded mouse HSCs successfully engrafted in SCID mice. PFT-α did not exhibit overt cytotoxic effects on human umbilical vein endothelial cells, human dermal fibroblasts, or MSCs under the experimental conditions. Conclusion: PFT-α significantly enhanced the ex vivo expansion of HSCs and appeared to preserve early engraftment potential. These findings indicate that PFT-α may represent a promising candidate for enhancing ex vivo hematopoietic stem cell expansion for therapeutic applications. Further studies should investigate long-term repopulation ability, multilineage differentiation potential, optimized dosing regimens, and efficacy in disease-specific or transplantation models.

Crimean–Congo hemorrhagic fever virus (CCHFV) remains a major public health threat due to its high mortality rates and the absence of approved antiviral therapies. The viral ovarian tumor (OTU) protease is a critical virulence factor that suppresses host innate immunity through its deubiquitinase activity, making it an attractive therapeutic target. In this study, we employed a structure-guided approach to identify and validate novel small-molecule inhibitors targeting the non-catalytic Y89-W99 pocket of the OTU protease. Recombinant OTU protease was successfully expressed, purified, and refolded, yielding a soluble and enzymatically active protein. Cellular assays confirmed that the enzyme retains robust deubiquitinase activity, significantly reducing global ubiquitin conjugates in mammalian cells. In silico analysis of a putative DUB inhibitor library identified several candidate inhibitors with favorable binding interactions within the Y89-W99 pocket. Biochemical validation using a fluorometric Ub-AMC assay revealed that multiple small molecules strongly inhibit OTU activity, including OTUi-10 (~93% inhibition), OTUi-13 (~87%), OTUi-1 (~85%), OTUi-4 and OTUi-11 (~81%), and OTUi-9 (~76%). Additional moderate inhibitors included OTUi-12 (~67%), OTUi-19 and OTUi-21 (~66%), and OTUi-5 (~57%). In silico drug-likeness and toxicity profiling filtered the library to four fully compliant candidates, OTUi-4, OTUi-10, OTUi-11, and OTUi-12, all free of predicted toxicity alerts. These findings suggest that the Y89–W99 pocket may be a pharmacologically relevant site worthy of further investigation and identify OTUi-10, OTUi-4, and OTUi-11 as promising preliminary hit compounds. The results also provide initial insights that may guide future optimization and mechanistic studies of OTU protease inhibitors targeting CCHFV.

Neospora caninum is a major apicomplexan pathogen responsible for significant reproductive losses in livestock, yet lacks effective therapeutics. Here, we identify and functionally characterize a previously unstudied OTU-like deubiquitinase (ncOTU; XP_003886403) as a key modulator of host–pathogen interactions. Sequence and structural analyses revealed conservation of the catalytic triad (D257, C260, H362) and a Y305-W315-G316 inhibition pocket analogous to viral OTU proteases. Recombinant ncOTU exhibited robust deubiquitinase activity and significantly reduced global ubiquitination levels in mammalian cells, preferentially targeting mono-ubiquitinated and low-molecular-weight substrates. Transcriptomic analysis demonstrated that ncOTU expression correlates with suppressed NF-κB signaling, type I interferon responses, and downstream antiviral effectors, while partially uncoupling upstream nucleic acid sensing pathways. Structure-based virtual screening and biochemical validation identified multiple small-molecule inhibitors targeting the conserved inhibition pocket. Dose-response analysis revealed submicromolar potency for ncOTUi-9 (IC₅₀ = 0.1 μM), ncOTUi-8 (0.2 μM), and ncOTUi-19 (0.3 μM), whereas ncOTUi-3 showed lower activity (7.1 μM). Interaction analyses confirmed stable binding within the inhibition pocket, with more extensive contact networks correlating with increased potency. Collectively, these findings establish ncOTU as a functional deubiquitinase that contributes to evasion and highlight it as a promising therapeutic target for neosporosis.

Babesia microti is the primary agent of human babesiosis, an emerging tick-borne disease with limited treatment options and growing evidence of drug resistance. Deubiquitinases (DUBs) play critical roles in protein homeostasis and host–pathogen interactions, yet none have been characterized in B. microti. Here, we report the first molecular cloning, expression, and functional characterization of an OTU-like cysteine protease from B. microti (bm-OTU). The recombinant bm-OTU protein (~22 kDa) was expressed in E. coli, purified to high homogeneity, and exhibited ~93% solubility under native conditions. In vitro fluorogenic assays confirmed its deubiquitinase activity. Expression of bm-OTU in HEK293T cells was associated with reduced ubiquitination in cells and increased apoptosis in this overexpression model, as demonstrated by flow cytometry and Western blot analyses. Furthermore, transcriptomic analysis revealed that bm-OTU modulates host immune pathways, notably suppressing the expression of interferon-stimulated genes (APOBEC3G, G1P2) while upregulating the pro-apoptotic gene BAK and the inflammasome sensor AIM2. These findings establish bm-OTU as a functional deubiquitinase and a potential virulence factor that may contribute to immune evasion and pathogenesis in babesiosis and may represent a potential target, pending further validation.

Background: MEIS proteins are essential homeobox transcription factors that play critical roles in development and have been increasingly implicated in oncogenesis, including breast cancer. Methods: In this study, we identified and characterized novel small-molecule MEIS2 inhibitors through in silico docking targeting the active region of the human MEIS2 homeobox domain. Lead candidates MEISi-2E, MEISi-3, and MEISi-4 were identified with binding energies ranging from −3.0 to −3.90 kcal/mol. The inhibitory potential of these molecules was validated in vitro using a species-conserved MEIS-Luciferase Reporter construct containing the TGACAG targeted locus. Results: Our results demonstrate that MEISi-2E, MEISi-3, and MEISi-4 significantly suppress MEIS-driven luciferase activity and downregulate the expression of Meis1, Meis2, and downstream genes such as IL17RB, CDH1, EGR2, PAX6, and SERPINE1 while upregulating negative regulator TGIF1 and SOX3. In breast cancer cell lines, these inhibitors exhibited potent growth inhibition, with MEISi-3 showing an exceptional IC50 as low as 0.1 μM in SK-BR-3 cells. Mechanistic studies using flow cytometry revealed that these inhibitors induce dose-dependent apoptosis and necrosis. Importantly, the novel inhibitors showed minimal toxicity to healthy human dermal and MRC5 fibroblasts, suggesting a favorable safety profile. Conclusions: These findings establish these small molecules as promising therapeutic candidates for targeting MEIS2-dependent pathways in breast cancer.

Background/Objectives: Hematopoietic stem cells (HSCs) sustain lifelong blood cell production and hold therapeutic promise, yet their ex vivo expansion remains constrained by an incomplete understanding of the metabolic and cellular mechanisms governing self-renewal. In this study, we investigated whether boron compounds boric acid (BA), sodium pentaborate pentahydrate (NaB), and sodium 2-pentaborate pentahydrate-8 (Na2B8) can promote the expansion of mouse HSCs by modulating key stem cell populations linked to metabolic fitness. Methods: Lineage-negative (Lin−) cells were magnetically isolated and treated with each boron compound for four days, followed by flow cytometric analysis of c-Kit, Sca-1, Lin-c-Kit+Sca-1+ (LSK), and LSKCD34^Low HSC-enriched subsets. Results: Our results show that boron derivatives exert distinct effects on these cellular markers. Notably, NaB treatment significantly increased the Lin-Sca-1+ cell fraction, while Na2B8 elevated both LSK and LSKCD34^Low ratios. Furthermore, the BA+NaB combination produced a statistically significant proliferative effect on Sca-1+ and c-Kit+ (CD117) cells. Conclusions: These findings indicate that specific boron compounds enhance ex vivo HSC expansion through yet-to-be-defined mechanisms that underpin HSC self-renewal. Further mechanistic studies are warranted to delineate the precise metabolic targets, but these results highlight boron compounds as promising tools for improving HSC expansion strategies.

Ubiquitination is a key post-translational modification regulating cellular signaling and innate immunity, and its reversal by deubiquitinases (DUBs) represents a critical mechanism exploited by pathogens for immune evasion. While ovarian tumor (OTU) domain-containing DUBs are well characterized in viral systems, their roles in fungal pathogens remain largely unexplored. In this study, we investigated two putative OTU domain-containing proteins derived from the plant pathogenic fungi Melampsora larici-populina (MlpOTU, EGG09943.1) and Taphrina deformans (TdOTU, CCG84064.1). Recombinant MlpOTU and TdOTU proteins were successfully expressed and purified from E. coli and exhibited high solubility and proper folding. Functional analyses in HEK293T cells demonstrated that both proteins significantly reduce global ubiquitination levels, confirming their deubiquitinase activity in vivo. Despite this shared enzymatic function, the two proteins displayed markedly distinct effects on host immune gene expression. MlpOTU selectively suppressed key antiviral effectors, most notably MX1, suggesting a targeted immune evasion strategy. In contrast, TdOTU induced robust upregulation of multiple immune-related genes, including type I interferons, indicating a divergent role. Neither MlpOTU nor TdOTU triggered robust apoptosis, supporting their role as modulators of host signaling rather than cytotoxic effectors. Collectively, these findings provide the first functional evidence that fungal OTU domain-containing proteins act as active deubiquitinases and reveal distinct strategies by which plant pathogens may manipulate host immune responses. This study establishes fungal OTU domains as promising targets for antifungal intervention and broadens our understanding of cross-kingdom evasion mechanisms.

Homeobox protein MEIS2 has been strongly implicated in colorectal cancer (CRC) progression and metastatic potential, making its targeted inhibition a promising therapeutic strategy. However, recently developed MEIS inhibitors are limited by poor aqueous solubility, instability under physiological conditions, and insufficient intracellular accumulation, which restrict their clinical applicability. To overcome these challenges, a multifunctional hybrid nanoemulsome system was developed by integrating boron–silane-doped carbon dots (CDs) with chitosan via glutaraldehyde crosslinking, followed by emulsification with oleic acid and non-ionic surfactants (Span 80 and Tween 20/80) in the presence of a MEIS inhibitor (MEISi-2). The resulting composite exhibited high structural stability, excellent biocompatibility, and a drug encapsulation efficiency of 96.2%. Fourier-transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) analyses confirmed successful hybridization and the formation of nanoemulsions with an average particle size of approximately 320 nm following drug loading. The system demonstrated controlled drug release under physiological conditions. In vitro studies using HCT116 CRC and HaCaT healthy keratinocytes revealed effective cellular uptake and selective cytotoxicity. The intrinsic fluorescence properties of CDs enabled real-time monitoring of intracellular drug delivery via DAPI-channel imaging. Overall, this hybrid nanoemulsome platform provides a stable and efficient delivery system for MEIS inhibitors and represents a promising strategy for the treatment of CRC. Furthermore, this approach may be extended to other poorly soluble amphiphilic therapeutic agents.

MEIS1, a member of the TALE-type homeobox gene family, has emerged as a pivotal regulator of cardiomyocyte cell cycle arrest and represents a promising therapeutic target. Our study reveals that inhibition of MEIS1 using two novel small molecules, MEISi-1 and MEISi-2, significantly enhances neonatal cardiomyocyte proliferation and cytokinesis. Specifically, MEISi-1 and MEISi-2 increased the proportion of proliferating cardiomyocytes (Ph3+TnnT cells) upto 4.5 fold and the percentage of cytokinetic cardiomyocytes (AuroraB+TnnT cells) by 2 fold, compared to untreated controls. MEIS1 inhibition resulted in a notable downregulation of MEIS1 target genes and cyclin-dependent kinase inhibitors, demonstrating its effect on key regulatory pathways. Additionally, the culture and differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes were studied, with MEIS1 inhibition showing no adverse effects on cell viability. Extended treatment with MEIS inhibitors led to a substantial upregulation of critical cardiac-specific genes, including a 15-fold increase in the expression of Nkx2.5. This upregulation significantly impacted both cardiac mesoderm and cardiac progenitor cells. These findings underscore the potential of MEIS1 inhibitors as effective agents in enhancing cardiac regeneration and highlight their therapeutic promise in regenerative cardiology.

Diabetes Mellitus (DM), tüm dünyada insanları etkileyen en yaygın hastalıklardan biridir. Kandaki düşük insülin seviyeleri ve yüksek glikoz seviyeleri ile karakterizedir. DM'nin önemli bir tedavisi a-glikosidaz enziminin inhibisyonudur. Piperazin ve sülfonamid yapılarının çeşitli biyolojik aktiviteleri bilinmektedir. Bu çalışmada beş adet fenilsülfonil piperazin türevi sentezlenip enzim inhibisyon kapasiteleri değerlendirildi. Sentezlenen moleküller (1-5), a-glukosidaz enziminin iyi derecede inhibe ettiği görüldü. Bileşik 1, a-glukosidaz enzimi için en yüksek inhibisyon potansiyeline sahiptir. İnhibisyon yüzdesi (83,52±0,41), referans molekül olan quercetine (81,41±0,02) göre daha yüksektir. Olası protein-ligand etkileşimlerini belirlemek amacıyla a-glukosidaz enzimi için en güçlü bileşik 1 için silico moleküler yerleştirme çalışmaları yapıldı. Ayrıca kuantum mekanik ve elektronik özelliklerinin değerlendirilmesi için bir DFT çalışması yapılmıştır. Son olarak bileşiklerin ADME profilleri teorik olarak analiz edildi.