Biomedicine & Pharmacotherapy

Acute Myeloid Leukemia (AML) is a life-threatening hematologic malignancy. Despite recent therapeutic advances, rising incidence rates emphasize the urgent need for identification of new targets and therapies. Roles of interleukin receptor-associated kinases IRAK1/4 are emerging in hematologic and solid malignancies. In AML, IRAK4 mRNA is overexpressed at diagnosis, relapses, in residual disease, and in FLT3-ITD-mutant cells, MDS, MPN, and MDS/MPN-negative subtypes. Compared with hematopoietic stem cells, IRAK4 is elevated in t(15;17), inv(16)/t(16;16), and t(11q23)/MLL subtypes, correlating with poor survival. Here, we disclose anti-AML activity of PSP-0119, a novel IRAK4 PROTAC degrader. PSP-0119, inhibited IRAK4 kinase activity, NF-{kappa}{beta} activity, and IL-1{beta}-induced IRAK4 phosphorylation. In-silico docking revealed interactions in CRBN/IRAK4/PSP-0119 ternary complex. PSP-0119 degraded IRAK4 in FLT3-mutant AML cell lines sparing FLT3-wild-type AML cells, FLT3-wild-type patient samples, and normal bone-marrow. Bulk-seq of PSP-0119 treated MOLM-13 cells revealed downregulation of eNOS, a poor AML prognosticator. PSP-0119 suppressed colony formation, cell viability, and MOLM-13 xenograft growth, and synergized with IRAK1 covalent inhibitor JH-X-119-01. PSP-0119 is metabolically stable, retaining 71% of parent compound at 60 minutes in human liver microsomes. In summary, IRAK4 degradation via PSP-0119 as a promising therapeutic strategy for treatment of FLT3-mutant AML.

Blood-brain barrier (BBB) contributes to maintenance of brain homeostasis. Gut microbiome composition affected BBB development and expression of tight junction proteins in rodents. However, we still do not know if theres any direct effect of gut microbiol composition on BBB permeability and function in normal adult animals. In current study, we determined temporal and spatial change of BBB permeability in rhesus monkeys receiving either oral or intravenous amoxicillin-clavulanic acid (AC), by monitoring CSF/serum albumin ratio (AR) and the volume transfer constant (Ktrans). We showed that oral, but not intravenous AC led to a significant alteration in gut microbiol composition and increase of BBB permeability in all monkeys, especially in thalamus area. Acetic acid and propionic acid might play a pivotal role in this newly found communication between gut and central nervous system. Antibiotics-induced gut microbiol composition change, especially the decreasing of acetic and propionic acid producing phyla and genera, leads to increase of BBB permeability, which may contribute to a variety of neurological and psychological diseases.

ObjectiveTranslocation of gut bacterial lipopolysaccharide (LPS) is associated with complications of cirrhosis. Current strategies to target bacterial translocation are limited to antibiotics with risk of resistance. This study aims to explore therapeutic potential of a non-absorbable, engineered carbon bead, Yaq-001 in cirrhosis and acute-on-chronic liver failure (ACLF) models. DesignThe performance of Yaq-001 was evaluated in in vitro studies. Two-rodent models of cirrhosis (4-week, bile duct ligation (BDL): Sham (n=36); Sham-Yaq-001 (n=30); BDL (n=37); BDL-Yaq-001 (n=44)) and ACLF (BDL-LPS: Sham-LPS (n=9); Sham-LPS-Yaq-001 (n=10); BDL-LPS (n=16); BDL-LPS-Yaq-001(n=12)). The treated-groups received Yaq-001 for 2-weeks. Samples were collected for assessment of organ and immune function, transcriptomics, microbiome composition and metabolomics. ResultsIn vitro, Yaq-001 exhibited rapid adsorption kinetics for endotoxin and bile acids without exerting an antibiotic effect. In vivo, Yaq-001 produced significant improvement in ALT, ammonia, liver cell death, portal pressure, markers of systemic inflammation and renal function in BDL animals. Yaq-001-treated ACLF animals had significantly better survival, ALT, portal pressure, brain water and creatinine. Ex-vivo LPS-induced reactive oxygen species production in portal venous monocytes and Kupffer cell populations was diminished with Yaq-001 treatment. Transcriptome analysis demonstrated a significant modulation of inflammation, cell death and senescence pathways in the liver, kidneys, brain and colon of Yaq-001-treated BDL rats. Yaq-001 impacted positively on the microbiome composition with significant modulation of Family Porphyromonadaceae and Genus Barnesiella. Urinary 1HNMR analysis suggested a shift in metabolomic signature in Yaq-001-treated BDL rats. ConclusionsThis study provides strong pre-clinical rationale for developing Yaq-001 for treatment of patients with cirrhosis. Significance of this studyO_ST_ABSWhat is already known on this topic?C_ST_ABSCurrent strategies to target bacterial translocation in cirrhosis are limited to antibiotics with risk of resistance. Yaq-001 is an insoluble, non-absorbable, non-antibiotic, engineered carbon bead of tailored porosities, which works as an adsorbent in the gut and is completely excreted after oral administration. What this study adds?O_LIYaq-001 rapidly adsorbs endotoxin, ammonia and bile acids without influencing bacterial growth kinetics in vitro. C_LIO_LIYaq-001 reduces mortality of ACLF animals and impacts positively on markers of gut permeability, liver injury, portal pressure, brain and kidneys in rodent models of cirrhosis and ACLF. C_LIO_LIYaq-001 administration was associated with positive impact on the composition of the gut microbiota, reduction in severity of endotoxemia and ammonia, which significantly reduced the severity of inflammation, cell death, signaling pathways and LPS sensitivity. C_LI How this study might affect research, practice or policy?The data provide the pre-clinical rationale to proceed to clinical trials in patients with cirrhosis aiming to prevent the occurrence of complications.

Epilepsies are a common and severe neurological condition characterized by spontaneous and recurrent seizures. Although anti-seizure medications are effective for most patients, about 30% remain pharmacoresistant. Moreover, uncontrolled seizures are associated with risk factors and shortened life expectancy for individuals with refractory epilepsy. Preclinical studies are an essential step for drug discovery and the zebrafish (Danio rerio) has been successfully employed for this purpose. In this study, we applied the zebrafish PTZ-seizure model to investigate the effect of two compounds on seizure suppression, Tripeptide (p-BTX-I) and the Cx43 peptide CX2. Zebrafish larvae at 6 days post-fertilization (dpf) were exposed to both compounds, according to their group, 24h prior to PTZ-seizure induction. We quantified the compounds effect on seizure latency, number of seizures and transcript levels of genes related to inflammation, oxidative stress, and apoptosis (il1b, tnfa, cox1, cox2a, il6, casp3a, casp9, baxa, bcl2a, nox1, sod1 and cat). Our results showed that CX2 at a concentration of 0.1 M/mL yielded the best outcome for seizure suppression as it reduced the number of seizures and increased the seizure latency. Additionally, CX2 treatment before PTZ-induced seizures decreased the transcript of il1b, il6, tnfa and cox1 genes, all related to inflammation. A bio-distribution study showed that the CX2 reached the zebrafish brain at both times investigated, 1h and 6h. Similarly, the tripeptide exhibited anti-inflammatory and anti-apoptotic action, reducing mRNA expression of the il1b and casp9 genes. Our findings suggest that both Tripeptide and CX2 hold translational potential for seizure suppression.

IntroductionTransient receptor potential ankyrin-1 positive (TRPA1+ve) nociceptors, primarily present as peptidergic neuronal afferents in the colon are sensors of disturbance in lower gastrointestinal tract including pain induced by different pathologies. Their therapeutic role in the alleviation of chronic pain (receptor antagonism and receptor desensitization) associated with inflammatory bowel diseases (IBD) is reported. However, there is limited literature available about their role in formation and sustenance of the mucosal layer, and its interaction with host physiology as well as luminal microbial community. The aim of this study focuses on the effects of nociceptive TRPA1 channel desensitization on colonic mucus production and gut health. MethodsTRPA1+ve nociceptors were desensitized by rectal administration of capsazepine. Ileum, colon was harvested and cecum content was collected. We performed morphological/histological analysis, gut permeability alteration, gene expression changes, colon metabolite profiling, and gut microbial abundance in these animals. ResultsWe found that presence of TRPA1-positive nociceptors is required for mucus layer integrity, using an intra-rectal capsazepine-induced TRPA1 desensitization model. Desensitization of TRPA1 positive nociceptors resulted in damaged mucosal lining, resultant increase in gut permeability and altered transcriptional profile of genes for goblet cell markers, mucus regulation, immune response and tight junction proteins. The damage to mucosal lining prevented its role in enterosyne (short chain fatty acids) actions. ConclusionThese results suggest that caution may need to be exercised before employing TRPA1 desensitization as a therapeutic option to alleviate pain caused due to IBD.

Graft-versus-host disease (GvHD) remains one of the major complications following allogeneic hematopoietic cell transplantation (allo-HCT), resulting in reduced quality of life, morbidity, and mortality in transplanted patients. Clinical strategies to prevent GvHD are frequently associated with off-target effects and dose-related toxicity. Given that oxidative stress is elevated in allo-HCT recipients and contributes to the pathogenesis of GvHD, the current study aims to characterize the role of N-acetylcysteine amide (NACA), a novel antioxidant, as the prophylactic treatment for acute GvHD. Using a murine GvHD model, we found that oral administration of NACA significantly reduced GvHD severity, prolonged survival, and improved the clinical manifestations and integrity of target organs compared to saline or N-acetylcysteine (NAC) treatment. NACA modulated splenic T cells differentiation with an increase in the regulatory (Treg) subset and a decrease in the cytotoxic (CD8+) subset. Moreover, inflammatory mediators, such as ROS and pro-inflammatory cytokines were downregulated by NACA treatment. In addition, NACA hindered donor T-cell proliferation in the recipients, and restrained Th1 and Th17, but not Th2 polarization. Importantly, NACA did not influence full donor engraftment in bone marrow and spleen. Taken together, our findings provide a new candidate for GvHD prophylactic treatment by targeting oxidative stress that can be easily translated to clinical use. Key PointsO_LINACA provides superior prophylactic effect against aGvHD compared to NAC in an allogeneic transplantation mouse model. C_LIO_LINACA treatment neither showed systemic toxicity nor altered the engraftment of the donor cells. C_LI

Doxorubicin (DOX) treatment increases the risk of myocardial dysfunction and heart failure, in which oxidative stress plays a central role. Empagliflozin (EMPA) has been shown to benefit heart failure patients, yet its underlying mechanism remains unclear. In this study, we showed that EMPA treatment improved cardiac function, ameliorated structural remodeling, and increased survival in DOX-treated mice. Furthermore, EMPA reduced DOX-induced reactive oxygen species (ROS) overproduction and myocardial injury. Through functional analysis overlapping DIC- and EMPA-regulated genes, we identified the involvement of JNK signaling in addition to redox pathways. Subsequent investigations demonstrated that EMPA restored redox homeostasis by inhibiting JNK activation, reactivating NRF2 and its downstream antioxidant proteins HO-1, NQO1, GPX4, and SOD2 both in vivo and in vitro. These protective effects were abolished by the Nrf2 inhibitor ML385 or the JNK activator anisomycin. Collectively, our findings indicate that EMPA protects against DOX-induced cardiotoxicity by modulating the JNK/Nrf2 signaling and attenuating oxidative stress. This study provides a rationale for further investigation of EMPA as a potential therapeutic strategy for chemotherapy-associated cardiac injury. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/697595v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@16743d4org.highwire.dtl.DTLVardef@96495borg.highwire.dtl.DTLVardef@aa343dorg.highwire.dtl.DTLVardef@4ba18c_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundNeuroinflammation driven by dysregulated adaptive and innate immune responses plays a central role in the pathogenesis of multiple sclerosis and related autoimmune disorders of the central nervous system. While bile acids are increasingly recognized as endogenous immunomodulators, their therapeutic exploitation has been limited by modest potency and incomplete mechanistic understanding. Here, we report the rational engineering of a bile acid-derived scaffold that yields a potent small molecule immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis, a preclinical model of multiple sclerosis. MethodsA focused series of bile acid-based compounds was established, leading to the identification of an androstane-triol derivative, BA59. The immunomodulatory activity of BA59 was evaluated using in vitro T cell differentiation assays, antigen-presenting cell phenotyping, and ex vivo immune profiling. Therapeutic efficacy was assessed in mice with established experimental autoimmune encephalomyelitis. Flow cytometry was used to characterize peripheral and central nervous system immune populations, including effector T cells, regulatory T cells, and antigen-presenting cells. Disease progression was monitored using clinical scoring and cumulative disease burden analyses. ResultsBA59 treatment significantly attenuated disease severity and cumulative disease burden when administered therapeutically after disease onset. Immunophenotyping revealed a reduction in pro-inflammatory T helper 17 cells accompanied by an increase in regulatory T cells expressing the ectonucleotidase CD39. BA59 also reprogrammed antigen-presenting cells toward a tolerogenic phenotype, characterized by enhanced programmed death-ligand 1 expression. These immune changes were observed both in peripheral lymphoid tissues and within the central nervous system. Importantly, BA59 did not induce broad immunosuppression but instead reshaped immune checkpoint signaling and regulatory pathways associated with neuroinflammatory resolution. ConclusionsThis study identifies BA59 as a first-in-class androstane-triol immunomodulator that ameliorates experimental autoimmune encephalomyelitis through coordinated regulation of T cell balance, immune checkpoints, and antigen-presenting cell function. Our findings highlight bile acid scaffold engineering as a viable strategy for developing small molecule therapeutics that reprogram neuroinflammatory immune circuits, offering a promising translational approach for multiple sclerosis and related neuroinflammatory diseases.

BackgroundMetabolic-dysfunction associated steatotic liver disease(MASLD) is a global epidemic affecting >30 % global population with no approved therapies till date. Recent reports suggest that activation of NLRP3 inflammasome may be involved in the pathogenesis of MASLD. We, therefore, aimed to identify small molecule inhibitor(s) of NLRP3 inflammasome as a potential therapeutic strategy to manage MASLD and validate their efficacy using in vitro and in vivo models of MASLD. MethodologyWe screened an in-house library of natural products using an in vitro phenotypic assay and identified a gut microbiota derived metabolite of dietary Tryptophan; indole-3 acetic acid (I3A) for its ability to inhibit the levels of IL-1{beta} and IL-18, which are elevated as a result of activation of NLRP3 inflammasome in differentiated THP1 cells. Subsequently, we carried out several in vitro and in vivo studies to confirm the mechanism of action of I3A and its ability to mitigate the key hallmarks of MASLD ResultsOur in vitro data suggest that I3A is an inhibitor of NLRP3 inflammasome. I3A was also found to improve the blood glucose level, plasma lipid profile, hepatic fat, and liver function in high-fat-high-fructose diet induced model of MASLD using C57BL/6 mice. ConclusionOur results show that I3A, which has been previously reported to be a gut microbiota-derived metabolite of dietary tryptophan, mitigates the key hallmarks of MASLD in an NLRP3 dependent manner. A dedicated structure-activity-relationship (SAR) study around the I3A chemotype may be carried out in future to identify novel NLRP3 inhibitors with desirable pharmacological profile.

Oncogenic metabolism depends on multifaceted mechanisms, including bidirectional inter-organelle communication between mitochondria and the nucleus, facilitating cellular adaptation at the transcriptomic, proteomic, and metabolomic levels. The mitochondrial protein complex composed of apoptosis-inducing factor (AIF) and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is essential for this mitochondrio-nuclear communication. The AIF/CHCHD4 complex mediates the mitochondrial import of cysteine-enriched nuclear gene-encoded proteins, thereby adapting the mitochondrial proteome to cellular energy demands. We report the discovery of M30-E05, an engineered flavonoid that binds to the NADH pocket of AIF, preventing its dimerization and disrupting the AIF/CHCHD4 complex. Molecular docking and gel electrophoresis analysis of mitochondrial AIF/CHCHD4 substrates expression confirm this mechanism. In cancer cells, M30-E05 reduces the expression of nuclear gene-encoded mitochondrial proteins such as AIF, CHCHD4, COX17, and MICU1. In addition, M30-E05 fragments the mitochondrial network and impairs mitochondrial respiration, causing profound alterations, particularly in lipid and aminoacid metabolism, as revealed by kinetic measurements of oxygen consumption and mass spectrometric metabolomics. Importantly, M30-E05 significantly reduces the viability of a human adult and pediatric osteosarcoma cancer cell panel, including those from patient-derived xenografts (PDX) of osteosarcomas, and induces apoptosis. When orally administered for two weeks to immunodeficient NSG mice, M30-E05 inhibited tumor growth in a subcutaneous PDX xenograft model without apparent toxicity. We anticipate that M30-E05, as a first-in-class metabolic inhibitor, could serve as the lead compound for a new class of targeted antineoplastic agents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/642976v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1b1add9org.highwire.dtl.DTLVardef@cac7corg.highwire.dtl.DTLVardef@101c9corg.highwire.dtl.DTLVardef@1c6845f_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundPreviously, it has reported that Peucedanol (PEU) possesses anti-bacterial activity. However, its effect and mechanism against inflammation remains unclear. MethodsIsothermal titration calorimetry (ITC) was used to assess binding affinities of PEU to pathogen associated molecular patterns (PAMPs) Kdo2-Lipid A (KLA), oligodeoxynucleotide 1826 (ODN 1826), and peptidoglycan (PGN). A lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation model and a cecum ligation and a puncture (CLP)-induced mouse sepsis model were used to assess efficacy and mechanism of PEU in vitro and in vivo. 16S ribosomal RNA (16S rRNA) sequencing was used to assay characteristics of intestinal flora of the sepsis mice. ResultsPEU had a moderate binding to KLA and ODN 1826. PEU significantly reduced supernatant tumor necrosis factor (TNF-) and interleukin 6 (IL-6), and downregulated protein expressions of toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 8 (MyD88), and nuclear factor kappa-B (NF-{kappa}B) in the LPS-treated cells. PEU remarkably increased the survival rate, reduced the serum TNF- and IL-6 levels, attenuated the CLP-induced pathological damage of intestine, increased proliferation-related proteins Bmi1 and Lgr5. Further, the anti-inflammatory effects of PEU were not significantly abolished in the present of chloroquine (CQ). Meanwhile, PEU significantly increased Chao1 index of the intestinal flora at the early stage of sepsis. In addition, PEU significantly changed composition of the flora at both phylum and genus levels. Moreover, PEU significantly affected metabolism-related pathways such as tricarboxylic acid (TCA) cycle, fatty acid degradation, secondary bile acid biosynthesis, and others. ConclusionsTaken together, PEU significantly inhibits LPS-induced inflammation in vitro and CLP-induced sepsis in vivo. Further, its anti-inflammatory effect is independent of the TLR4/myD88/NF-{kappa}B pathway. In addition, PEU improves the intestinal flora imbalance at the early stage of sepsis.

Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. In this study, we hypothesized that pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, we designed a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles that enabled us to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibited negligible dopamine D2 and D3 receptor binding, unlike loxapine. We found that compound 6 displayed potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.

BackgroundCircadian rhythm disruption (CRD) is a risk factor for irritable bowel syndrome (IBS), but the mechanism linking CRD to intestinal barrier dysfunction remains unclear. This preclinical study aimed to clarify whether CRD impairs intestinal barrier integrity via gut microbiota dysbiosis and the "apoptosis-inflammation-oxidative stress" cascade. MethodsTwenty-four male C57BL/6 mice were randomized into control (12h light/dark, n=12) and CRD (21-day continuous light, n=12) groups. Circadian disruption was verified via locomotor activity, serum melatonin/serotonin, and clock gene expression. Intestinal barrier function, microbiota, apoptosis, inflammation, and oxidative stress were assessed using FITC-dextran permeability, 16S rRNA sequencing, Western blotting (WB), TUNEL, and ELISA. ResultsCRD increased intestinal permeability (+114.7%, p<0.001), shortened villi (-25.6%, p=0.018), downregulated tight junction proteins (ZO-1, Occludin, p<0.05), and altered microbiota (family-level: decreased Prevotellaceae, increased Bacteroidaceae, p<0.05). It also activated the apoptosis-inflammation-oxidative stress cascade (Caspase-3/{beta}-actin: +1.4-fold, IL-1{beta}: +44.1%, MDA: +50%, CAT: -90%, all p<0.05). ConclusionsCRD impairs intestinal barrier integrity via gut microbiota dysbiosis and the apoptosis-inflammation-oxidative stress cascade. These preclinical findings identify gut microbiota and the apoptosis-inflammation-oxidative stress cascade as potential targets for further investigating IBS associated with CRD.

Triglyceride rich macrophages (foam cells) are a hallmark of necrotic granulomas in tuberculosis, and multiple antimicrobial functions are down-regulated in these cells. In this study, we assessed the ability of two different compounds to reduce triglyceride content and intracellular burden in Mycobacterium tuberculosis (Mtb)-infected macrophages: A-922500 (DGATi), an inhibitor of diacylglycerol acyltransferase 1, an enzyme involved in triglyceride synthesis; and LY2584702 (p70S6Ki), an inhibitor of p70 S6 kinase, a serine/threonine kinase involved in mTORC-1dependent lipid biogenesis. Additionally, we evaluated the adjunctive activity of these inhibitors as host-directed therapies against chronic Mtb infection in C3HeB/FeJ mice. DGATi and p70S6Ki significantly reduced the lipid content and bacillary burden in Mtb-infected human monocyte-derived macrophages. In Mtb-infected mice, each inhibitor reduced the triglyceride content (P[&le;] 0.0001) in cells from bronchoalveolar lavage samples. Adjunctive treatment of DGATi with isoniazid and p70S6Ki monotherapy reduced the lipid droplet content (P[&le;] 0.05) within lung macrophages of Mtb-infected mice. However, neither inhibitor reduced the lung bacterial burden in Mtb-infected mice alone or in combination with isoniazid, and they did not alter lung inflammation. These findings provide further insights into the role of foam cells in tuberculosis pathogenesis and the utility of interventions targeting these cell populations as adjunctive host-directed therapies.

The microbiome-gut-brain axis has been implicated in multiple bodily systems and pathologies, and intentional manipulation of the gut-microbiome has yielded clinically significant results. Here, we examined the effects of bi-directional fecal microbial transplants (FMT) between methylone-induced hyperthermic tolerant (MHT) and methylone-naive (MN) rats. Rats treated with methylone once per week developed tolerance to methylone-induced hyperthermia by the fourth week. Once tolerant, daily bi-directional FMT between the two groups were performed for seven days prior to the next methylone treatment. The FMT abated the developed tolerance in the MHT group. When treated with methylone for the first time following FMT, recipient MN rats displayed significant tolerance to hyperthermia despite it being their initial drug treatment. Post-FMT, MHT rats displayed elevations in norepinephrine and expression of UCP1, UCP3 and TGR5 in brown adipose tissue, with reductions in expression of TGR5 and UCP3 in skeletal muscle. The pre- and post-FMT methylone tolerance phenotypes of transplant recipients are concurrent with changes in the relative abundance of several Classes of Proteobacteria, most evident for Gammaproteobacter and Alphaproteobacter. MHT recipients demonstrated a marked increase in the relative proportion of the Firmicutes Class Erysipelotrichia. These findings suggest that transplantation of gut-microbiomes can confer phenotypic responses to a drug.

Acute myeloid leukaemia (AML) is a haematopoietic malignancy comprising different genetic subtypes with a common hallmark of differentiation arrest. In abnormal haematopoiesis, overcoming the differentiation blockade has emerged as an attractive therapeutic strategy. In a screen with genetically distinct AML cell lines, histone deacetylase inhibitors (HDACis) were observed to cause an upregulation in the expression of CD11b, a myeloid differentiation marker. These caused changes in cell morphology, block in proliferation, and cell cycle arrest at the G1 phase. To gain insights onto the mechanism of these compounds, we planned to prepare inactive probes devoid of the zinc binding motif. However, these compounds were unexpectedly still able to initiate differentiation, albeit through a distinct target and via a G2 arrest. Subsequent RNA sequencing studies supported the differentiation phenotype for the HDACis and highlighted the role of cell cycle regulatory kinases for the effect observed in the probe molecules. We then showed that these inhibit Aurora A and GSK3 kinases, suggesting their potential as therapeutic targets for differentiation therapy in AML. Our work supports the importance of properly validating inactive tool compounds and their potential to identify novel targets.

LEAF-4L6715 is a liposomal formulation encapsulating transcrocetin (TC) developed to enhance the diffusion of oxygen in the body. Here, we report the final results of the phase I/II clinical trial (NCT04378920; EUDRACT2020-001393-30) initiated to identify an optimal regimen and to assess the activity of TC in the context of acute respiratory distress syndrome (ARDS). More specifically, LEAF-4L6715 was developed to treat patients with ARDS due to severe SARS-CoV-2 infection who have a ratio of partial arterial pressure to inspired fraction of oxygen (PaO2/FiO2 ratio) <200 treated with artificial ventilation support in an intensive care unit. A total of 37 patients were treated (across 6 dosing cohorts) with LEAF-4L6715 given as an intravenous infusion for over 90 minutes. The dose of LEAF-4L6715 was increased until the transaminase levels were elevated and 4 grade 3 events occurred among 8 patients. The recommended dosage was determined to be a fixed concentration of 300 mg administered every 12 hours. An improvement in the PaO2/FiO2 ratio and SOFA score was observed. The overall 28-day survival rate of 81%. This study identified the recommended dose for LEAF 4L6715 and the dose-limiting toxicity and showed an overall favorable risk/benefit profile. These preliminary findings are promising for the activity of LEAF-4L6715 but will require confirmation in a randomized phase III trial.

The experimental high-throughput screening (HTS) methods, exemplified by CRISPR- based screening techniques, have revolutionized target identification in drug discovery. However, such screens frequently yield extensive, often unrelated target lists necessitating costly and time-intensive experimental evaluation and validation. To address this challenge, we propose a dual-filter strategy that integrates literature-mined targets with CRISPR/Cas9 screening outputs, systematically prioritizing the most credible candidates and thereby reducing the experimental validation burden and increasing success rate. To validate this strategy, we applied it with hand-foot syndrome (HFS), a clinically challenging side effect induced by fluoropyrimidine treatment. We identified ATF4 as a key regulator of 5-fluorouracil (5-FU) toxicity in the skin and revealed forskolin as a potential therapeutic agent of HFS through the strategy. Mechanistically, forskolin triggers MEK/ERK-dependent ATF4 induction, subsequently driving 5-FU detoxification via the ATF4-mediated eIF2/I{kappa}B signaling pathway. Our findings demonstrate that this dual-filter strategy could notably accelerate drug discovery by reducing experimental validation burden after target screening.

The efficacy of anthracycline-based chemotherapeutics, which include doxorubicin and its structural relatives daunorubicin and idarubicin, remains almost unmatched in oncology, despite a side effect profile including cumulative dose-dependent cardiotoxicity, therapy-related malignancies and infertility. Detoxification of anthracyclines while preserving their anti-neoplastic effects is arguably a major unmet need in modern oncology, as cardiovascular complications that limit anti-cancer treatment are now a leading cause of morbidity and mortality among the 17 million cancer survivors in the U.S.. To address this, we examined different clinically relevant anthracycline drugs with respect to a series of features including mode of action (chromatin and DNA damage), bio-distribution, anti-tumor efficacy and cardiotoxicity in pre-clinical models and patients. We show that different anthracycline drugs have surprisingly individual efficacy and toxicity profiles. In particular, aclarubicin stands out in pre-clinical models and clinical trials as it potently kills cancer cells, does not induce therapy-related malignancies or cardiotoxicity, and can be safely administered even after a maximum cumulative dose of either ida- or doxorubicin has been reached. Retrospective analysis of aclarubicin used in second-line treatment of relapsed/refractory AML patients showed similar survival effects to its use in first line, leading to an almost 25% increase in 5-year overall survival. Considering individual anthracyclines as different drugs provides new treatment options that strongly improve survival of cancer patients while limiting the toxic side-effects. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/23298950v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@4d6483org.highwire.dtl.DTLVardef@101d8b7org.highwire.dtl.DTLVardef@3adf1aorg.highwire.dtl.DTLVardef@10d2c49_HPS_FORMAT_FIGEXP M_FIG C_FIG

Recombinant granulocyte colony-stimulating factor (G-CSF) is the most commonly used agent for treating neutropenia and mobilizing hematopoietic stem cells (HSCs) for transplantation. However, some patients do not respond effectively to existing mobilization protocols. To address this, the development of new therapeutic approaches is necessary. One potential strategy is the pharmacological induction of endogenous mobilizing factors, which can be achieved through the administration of cobalt protoporphyrin IX (CoPP). CoPP induces mobilization of HSCs and granulocytes by increasing endogenous G-CSF production, though the optimal dosing and potential side effects remain unclear. The aim of our study was to optimize the dose and timing of CoPP administration and evaluate its safety in mobilizing cells from the bone marrow to the blood. Our results show that CoPP exerts a dose-dependent mobilizing effect, with the highest G-CSF levels and number of mobilized leukocytes observed in mice treated with 10 mg/kg of CoPP. While there were no severe adverse effects, there were mild fluctuations in markers of liver and kidney function, including a slight reduction in urea nitrogen (BUN) and glucose levels during the five days of administration. Additionally, although most parameters normalized within 30 days after treatment, the decrease in BUN persisted. Mice experienced short-term weight loss following CoPP administration, but they regained their initial weight within two weeks. By day 30, leukocyte counts, hematopoietic stem and progenitor cells (HSPCs) in bone marrow, and G-CSF concentration in the blood had returned to baseline. This study demonstrates that CoPP mobilizes cells from the bone marrow to the blood in a dose-dependent manner, with mild side effects, including temporary changes in biochemical markers and a sustained reduction in BUN levels.