Heparin-induced thrombocytopenia (HIT) is an immune prothrombotic disorder characterized by the binding of platelet-activating immunoglobulin G antibodies to platelet factor 4/heparin. In platelets, this leads to cross-linking of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor FcγRIIa, platelet activation, and thrombocytopenia, which in combination with extensive thrombin generation significantly increases the risk of thrombosis. Our laboratory has previously demonstrated that 12-lipoxygenase (12-LOX), an oxygenase primarily expressed in platelets, plays a critical role in platelet activation through FcγRIIa. In this study, we aimed to determine the effectiveness of VLX-1005, a potent and selective inhibitor of 12-LOX, alone or in combination with argatroban, in preventing HIT. Pretreatment with VLX-1005 attenuated aggregation of human washed platelets stimulated with a HIT immune complex in vitro. VLX-1005 prevented ITAM-induced human whole-blood impedance alone or in combination with argatroban. VLX-1005 treatment impaired platelet adhesion and accumulation on a collagen-coated surface under shear stress. Mice expressing transgenic human FcγRIIa and 12-LOX experienced severe thrombocytopenia and thrombosis after a HIT-like challenge, whereas mice expressing transgenic human FcγRIIa with 12-LOX knockout were completely refractory to HIT pathology. VLX-1005 treatment did not affect coagulation or increase the risk of bleeding. This study demonstrates that inhibition of 12-LOX might be an effective intervention for preventing ITAM-regulated platelet activation, such as in HIT, and is independent of argatroban effects in blood. Importantly, VLX-1005 prevents platelet activation and does not increase the bleeding risk associated with direct thrombin inhibitors, such as argatroban.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. The severe disease outcomes are often associated with a hyperinflammatory response known as a "cytokine storm." The mechanisms underlying this exaggerated immune response remain incompletely understood. This study aimed to investigate the molecular pathways contributing to the severe inflammatory damage and mortality associated with COVID-19. SARS-CoV-2 hijacks host lipid metabolism, particularly the phospholipase A2 (PLA2) pathway, leading to the production of bioactive lipid mediators, including 12-lipoxygenase (12-LOX)-derived lipid mediators in platelets, and in lung and vascular cells. We hypothesized that 12-LOX drives the hyperinflammatory response and disease severity, and that its inhibition could reduce inflammation and improve outcomes. Analysis of autopsy lung samples from COVID-19 decedents and SARS-CoV-2-infected K18-hACE2 transgenic mice revealed increased 12-LOX expression. We evaluated VLX-1005, a selective small-molecule 12-LOX inhibitor, in infected mice. Treatment initiated 48 h post-infection significantly improved survival, reduced body weight loss, and decreased lung inflammation compared to controls. Notably, male mice showed higher survival rates than females. VLX-1005 treatment also suppressed key chemokines and cytokines associated with the cytokine storm, and reduced lung damage. These findings identify 12-LOX as a critical mediator of the hyperinflammatory response in severe COVID-19 and support its inhibition as a promising therapeutic strategy to mitigate inflammatory damage and reduce mortality. IMPORTANCE:This study provides critical insights into the mechanisms underlying severe COVID-19, identifying 12-lipoxygenase (12-LOX) as a key driver of the hyperinflammatory response that contributes to disease severity and mortality. By demonstrating that SARS-CoV-2 hijacks host-lipid metabolism to elevate proinflammatory lipid mediators, the research uncovers a novel pathogenic pathway that exacerbates lung inflammation. The use of VLX-1005, a selective 12-LOX inhibitor, significantly improved survival and reduced inflammatory damage in a mouse model, highlighting its therapeutic potential. These findings not only deepen our understanding of COVID-19 pathogenesis but also position 12-LOX as a promising intervention target, offering a new avenue to mitigate the effects of cytokine storms in severe cases.

Basal breast cancer is a subtype with a poor prognosis in need of more effective therapeutic approaches. Here we describe a unique role for the KDM4C histone lysine demethylase in KDM4C-amplified basal breast cancers, where KDM4C inhibition reshapes chromatin and transcriptomic landscapes without substantial alterations of its canonical substrates, trimethylated histone H3 lysine 9 (H3K9me3) and lysine 36 (H3K36me3). Rather, KDM4C loss causes proteolytic cleavage of histone H3 mediated by cathepsin L (CTSL), resulting in decreased glutamate-cysteine ligase expression and increased reactive oxygen species. CTSL is recruited to the chromatin by the grainyhead-like 2 (GRHL2) transcription factor that is methylated at lysine 453 following KDM4C inhibition, triggering CTSL histone clipping activity. Deletion of CTSL rescued KDM4-loss-mediated tumor suppression. Our study reveals a function for KDM4C that connects cellular redox regulation and chromatin remodeling.

Obesity-associated inflammation is characterized by macrophage infiltration into peripheral tissues, contributing to the progression of prediabetes and type 2 diabetes. 12-lipoxygenase (12-LOX) catalyzes the formation of pro-inflammatory eicosanoids and promotes the migration of macrophages, yet its role in obesity-associated inflammation remains incompletely understood. Furthermore, differences between mouse and human orthologs of 12-LOX have limited efforts to study existing pharmacologic inhibitors of 12-LOX. In this study, we used a human gene replacement mouse model in which the gene encoding mouse 12-LOX (Alox15) is replaced by the human ALOX12 gene. As a model of obesity and dysglycemia, we administered male mice a high-fat diet. We subsequently investigated the effects of VLX-1005, a potent and selective small molecule inhibitor of human 12-LOX. Oral administration of VLX-1005 resulted in improved glucose homeostasis, decreased β-cell dedifferentiation, and reduced macrophage infiltration in islets and adipose tissue. Analysis of the stromal vascular fraction from adipose tissue showed a reduction in myeloid cells and cytokine expression with VLX-1005 treatment, indicating decreased adipose tissue inflammation. In a distinct mouse model in which Alox15 was selectively deleted in myeloid cells, we observed decreased β-cell dedifferentiation and reduced macrophage infiltration in both islets and adipose tissue, suggesting that the effects of VLX-1005 may relate to the inhibition of 12-LOX in macrophages. These findings highlight 12-LOX as a key factor in obesity-associated inflammation and suggest that 12-LOX inhibition could serve as a therapeutic strategy to improve glucose homeostasis and peripheral inflammation in the setting of obesity and type 2 diabetes.

Disclosure: K.B. Kaylan: None. T. Nargis: None. K. Figatner: None. J.E. Wang: None. S. Pratuangtham: None. A. Chakraborty: None. I. Casimiro: None. J.L. Nadler: None. M.B. Boxer: MBB is a Veralox Therapeutics employee. D.J. Maloney: DJM is a Veralox Therapeutics employee. R.M. Anderson: None. R.G. Mirmira: RGM received an investigator-initiated award from Veralox Therapeutics. RGM serves on the Scientific Advisory Board for Veralox Therapeutics. S.A. Tersey: SAT received an investigator-initiated award from Veralox Therapeutics. Individuals with obesity can develop peripheral insulin resistance and hyperinsulinism even without impaired glucose tolerance or type 2 diabetes (T2D). Obesity is also associated with macrophage infiltration in peripheral tissues, further contributing to insulin resistance and progression to T2D. The enzyme 12-lipoxygenase (12-LOX) catalyzes the formation of pro-inflammatory eicosanoids and is known to promote the migration of macrophages, yet its role in T2D and obesity-associated inflammation remains incompletely understood. Here, we used a human gene replacement mouse model in which endogenous mouse Alox15 is replaced by the human ALOX12 (B6.hALOX12). As a human-relevant model of obesity and T2D, we treated male B6.hALOX12 mice at 8 weeks of age with high-fat diet (HFD, 60% total calories from fat) and vehicle or VLX-1005, a potent and selective inhibitor of 12-LOX, given by oral gavage at 30 mg/kg/day for 10 weeks. B6.hALOX12 mice treated with VLX-1005 showed improved random-fed blood glucose levels and glucose tolerance testing with non-statistically significant improvements in insulin tolerance testing, glucose-stimulated insulin secretion, and homeostasis model assessment of insulin resistance values. In these mice, we additionally observed no changes in β cell mass, decreased β cell dedifferentiation, and decreased macrophage infiltration in islets and adipose tissue. Western blot analysis of adipose tissue immediately after insulin injection (3 units/kg) showed improved biochemical insulin sensitivity by phosphorylated AKT with VLX-1005 treatment. Isolation of the stromal vascular fraction from adipose tissue showed decreases in CD11b+ myeloid cells and Tnfα expression with VLX-1005 treatment. In a complementary transgenic zebrafish model in which macrophages are labelled by GFP, Tg(mpeg:eGFP)gI22, we observed decreased macrophage infiltration in pancreatic islets with VLX-1005 treatment after a HFD treatment (5% egg yolk). Last, to answer whether LOX enzyme expression has cell type-specific effects, we generated a conditional knockout of Alox15 in myeloid cells (B6.Alox15Δmyel). In B6.Alox15Δmyel mice, we observed unchanged glucose homeostasis and β cell mass, decreased β cell dedifferentiation, and decreased macrophage infiltration in both islets and adipose tissue. In summary, inhibition of 12-LOX with VLX-1005 in B6.hALOX12 mice results in improvement in glucose homeostasis and decreased macrophage infiltration in islets and adipose tissue. Our mouse model provides a platform by which to investigate the pathophysiological role of 12-LOX in other metabolic diseases, such as atherosclerosis and metabolic-dysfunction associated steatotic liver disease. These findings also suggest that 12-LOX inhibition could serve as a therapeutic strategy for obesity and T2D. Presentation: Monday, July 14, 2025

Introduction VLX-1005 is a novel small molecule inhibitor of platelet-type 12-lipoxygenase (12-LOX) intended for the prevention or treatment of thrombosis in patients with heparin-induced thrombocytopenia (HIT). Argatroban, a direct thrombin inhibitor, is currently the only agent approved in the U.S. for use in HIT. Its use is associated with a substantial risk of major bleeding in HIT patients, and a stubbornly high rate of thrombosis. As VLX-1005 and argatroban are co-administered in the ongoing VLX-1005-003 ALATHEA (A study of VLX-1005 to evaLuAte Thrombocyte change in HEpArin Induced Thrombocytopenia) clinical trial in HIT patients, the present study was designed to characterize the safety and tolerability of these agents alone and in combination, and the pharmacokinetics and pharmacodynamics and potential interaction of both agents in a population of healthy subjects. Methods This was a 3-period, open-label, Phase I, drug-drug interaction (DDI) study of VLX-1005 and argatroban, each administered intravenously via PICC line to healthy volunteers. Treatment A was argatroban (5 mcg/kg/min constant IV infusion for 6 hours without a loading dose). Treatment B was VLX-1005 (400 mg IV over 1 hour). Treatment C was argatroban at 5 mcg/kg/min constant IV infusion for 6 hours, VLX-1005 dosed at 400 mg IV over 60 minutes from 180 minutes to 240 minutes from the start of the argatroban infusion. Twelve (12) healthy male or female (non-lactating and not of childbearing potential) subjects aged 19 to 55 inclusive, were randomized to 1 of 3 treatment sequences: A-B-C or B-C-A, or C-A-B with a 1-week washout period between each period to avoid carryover effects. During each treatment period, monitoring of vital signs, laboratory tests (hematology, clinical biochemistry, and coagulation studies with monitoring of aPTT), and 12-lead ECG was conducted by appropriately trained personnel. Telemetry was performed for 24-hour time periods postdose in each treatment. PK samples were obtained in each treatment period, analyzed and PK parameters calculated. Results A total of 12 participants received study treatments, and 11 participants completed the full dosing period. One participant (Treatment Sequence A-B-C) was discontinued by the investigator on Day 3 of Period 2 due to behavioral issues and noncompliance with clinic rules. There were no discontinuations or study disruptions because of treatment. There were 12 participants included in the safety analyses and 11 participants included in the PK analyses. There were no deaths, SAEs, or participant discontinuations due to AEs in this study. The incidence of AE reporting was minimal and was observed to be similar across the 3 treatments. The most common event reported overall was vessel puncture site pain, reported by 3 [25%] participants, followed by vessel puncture site bruise and rhinorrhea (2 [17%] participants each). The investigator considered 18 of the 28 TEAEs to be mild (grade 1) and 10 to be moderate (grade 2); all events resolved. Coadministration of argatroban and VLX-1005 had no effect on the PK of either drug and no impact on the effect of argatroban on activated partial thromboplastin time (aPTT) in healthy human adult participants. Conclusions All treatment regimens appeared to be safe and well tolerated by the male and female healthy human subjects in this study; no evidence of increased bleeding was noted across treatment groups. Coadministration of argatroban and VLX-1005 had no effect on the PK of either drug. The administration of VLX-1005 had no effect on aPTT in healthy human adult subjects. The coadministration of VLX-1005 with argatroban had no impact on the effect of argatroban on aPTT in healthy human adult subjects. The Phase 2 ALATHEA (A study of VLX-1005 to evaLuAte Thrombocyte change in HEpArin Induced Thrombocytopenia) study of VLX-1005 in HIT is currently ongoing.

File contains synthesis of CN210, sequences for siRNAs, supplementary tables 1 to 3, supplementary figures 1 through 8 and figure legends.

PDF - 89K, Supplementary Materials and Methods.

PDF - 188K, Suface Plasmon Resonance (SPR) profiles binding for JLT048 and NCGC00183674 to recombinant TDP1 bound to the surface.

Abstract Disclosure: T. Nargis: None. A. Chakraborty: None. K. Figatner: None. D. Maloney: None. M. Boxer: None. S.A. Tersey: None. R.G. Mirmira: None. Type 1 diabetes (T1D) is an autoimmune disorder characterized by islet inflammation (insulitis). We have recently shown that a key mediator of inflammatory signaling in the islet beta cell is the enzyme 12-lipoxygenase (12-LOX), which generates proinflammatory eicosanoids that augment dysfunctional insulin secretion and beta-cell visibility to the immune system. Whereas inhibition of 12-LOX offers an opportunity to modify T1D progression by altering how the beta cell responds to insulitis, a barrier to testing inhibitors in preclinical models is the specificity of next generation inhibitors toward the human enzyme. To generate a preclinical platform for the study of human-specific 12-LOX inhibitors, we developed a mouse model in which the mouse-equivalent Alox15 coding region was replaced with the human ALOX12 gene, while retaining the mouse upstream control region. These mice (hALOX12) were developed on both the C57BL/6J and NOD genetic backgrounds, then subjected to treatment with the human 12-LOX inhibitor VLX-1005 to evaluate diabetes progression. C57BL/6J-wildtype and C57BL/6J-hALOX12 mice were confirmed to express exclusively mouse Alox15 and human ALOX12, respectively, using RNA isolated from islets. To test susceptibility to toxin-induced diabetes, C57BL/6J-wildtype and C57BL/6J-hALOX12 were subjected to multiple low-dose streptozotocin (STZ) to induce diabetes. Both wildtype and humanized mice exhibited gradual and equivalent hyperglycemia (within 25 days) following STZ injection. Following intraperitoneal VLX-1005 therapy (30 mg/kg), whereas C57BL/6J-wildtype mice developed diabetes (with a delay of 10 days) following STZ, C57BL/6J-hALOX12 mice showed complete protection against STZ-induced diabetes and exhibited significantly better glucose tolerance compared to vehicle- or VLX-1005-injected wildtype controls and vehicle-injected C57BL/6J-hALOX12 mice. Next, hALOX12 mice were backcrossed onto the NOD.ShiltJ background model of autoimmune T1D. NOD-hALOX12 mice received either vehicle or VLX-1005 (30 mg/kg) orally during the prediabetic phase (6-10 weeks of age) and were followed for diabetes incidence. Mice receiving VLX-1005 showed a significant delay with only 30% of female and 7% of male mice developing diabetes over 25 weeks of age. Pancreatic sections from the mice treated with VLX-1005 showed reduced insulitis and increased beta cell mass compared to vehicle controls. In summary, the hALOX12 mouse model serves as a preclinical translational platform to interrogate effects of next generation 12-LOX inhibitors and demonstrates the potential of VLX-1005 to modify T1D disease progression. Presentation: Saturday, June 17, 2023

PDF - 119K, Schematic representation of the AlphaScreen 3'-phospho-tyrosine DNA substrate used in the TDP1 qHTS assay.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a major concern for all individuals that undergo cardiac bypass surgeries or require prolonged heparin exposure. HIT is a life- and limb-threatening adverse drug reaction with an immune response following the formation of ultra-large immune complexes that drive platelet activation through the receptor FcγRIIA. Thrombotic events remain high following the standard of care treatment with anticoagulants, while increasing risk of bleeding complications. This study sought to investigate a novel approach to treatment of HIT. Recent reports demonstrate increased procoagulant activity in HIT; however, these reports required analysis ex vivo, and relevance in vivo remains unclear. METHODS: Using human and mouse model systems, we investigated the cooperativity of PARs (protease-activated receptors) and FcγRIIA in HIT. We challenged humanized FcγRIIA transgenic mice with or without endogenous mouse Par4 (denoted as IIA-Par4 +/+ or IIA-Par4 − /− , respectively) with a well-established model IgG immune complex (anti [α]-CD9). Furthermore, we assessed the procoagulant phenotype and efficacy to treat HIT utilizing inhibitor of 12-LOX (12[S]-lipoxygenase), VLX-1005, previously reported to decrease platelet activation downstream of FcγRIIA and PAR4, using the triple allele HIT mouse model. RESULTS: IIA-Par4 +/+ mice given αCD9 were severely thrombocytopenic, with extensive platelet-fibrin deposition in the lung. In contrast, IIA-Par4 −/− mice had negligible thrombocytopenia or pulmonary platelet-fibrin thrombi. We observed that pharmacological inhibition of 12-LOX resulted in a significant reduction in both platelet procoagulant phenotype ex vivo, and thrombocytopenia and thrombosis in our humanized mouse model of HIT in vivo. CONCLUSIONS: These data demonstrate for the first time the need for dual platelet receptor (PAR and FcγRIIA) stimulation for fibrin formation in HIT in vivo. These results extend our understanding of HIT pathophysiology and provide a scientific rationale for targeting the procoagulant phenotype as a possible therapeutic strategy in HIT.

Abstract Drug screening against novel targets is warranted to generate biochemical probes and new therapeutic drug leads. TDP1 and TDP2 are two DNA repair enzymes that have yet to be successfully targeted. TDP1 repairs topoisomerase I–, alkylation-, and chain terminator–induced DNA damage, whereas TDP2 repairs topoisomerase II–induced DNA damage. Here, we report the quantitative high-throughput screening (qHTS) of the NIH Molecular Libraries Small Molecule Repository using recombinant human TDP1. We also developed a secondary screening method using a multiple loading gel-based assay where recombinant TDP1 is replaced by whole cell extract (WCE) from genetically engineered DT40 cells. While developing this assay, we determined the importance of buffer conditions for testing TDP1, and most notably the possible interference of phosphate-based buffers. The high specificity of endogenous TDP1 in WCE allowed the evaluation of a large number of hits with up to 600 samples analyzed per gel via multiple loadings. The increased stringency of the WCE assay eliminated a large fraction of the initial hits collected from the qHTS. Finally, inclusion of a TDP2 counter-screening assay allowed the identification of two novel series of selective TDP1 inhibitors. Mol Cancer Ther; 13(8); 2116–26. ©2014 AACR.

Triple-negative breast cancer (TNBC) is a subtype accounting for 20-30% all breast cancer cases and is a highly aggressive disease with inferior prognosis. The acquired resistance is a major obstacle for efficient therapy and discovery of novel clinically-actionable targets has become an urgent need. In this study, we carried out an in-depth functional characterization of histone demethylase KDM4C in TNBC and uncovered a novel mechanism underlying KDM4C-driven tumorigenesis. KDM4C is the second most frequently amplified histone demethylase in TNBC. Genetic knockdown or pharmacological inhibition of KDM4C in two amplified basal breast cancer cell lines, SUM149 and HCC1954, drastically suppressed primary tumor growth in vitro and in vivo. Transcriptomic analysis underlined oxidative phosphorylation impairment as functional consequences of KDM4C blockade. Surprisingly, integrating series of histone ChIP-seq and ATAC-seq showed that KDM4C suppression caused accessible chromatin remodeling without substantial changes of its canonical substrates H3K9me3 and H3K36me3. Rather KDM4C loss caused proteolytic cleavage at histone 3 N-terminus (Ala21 site) identified by histone mass spectrometry. Protease inhibitor array pointed out cathepsin L (CTSL) as the endopeptidase mediating this procedure. KDM4C blockade induced CTSL activation and promoted H3 tail clipping at around 30% CTSL binding sites, which was associated with restricted chromatin accessibility and transcriptomic reprogramming. Proteomic interactome profiling revealed that grainyhead like transcription factor 2 (GRHL2) tightly binds to CTSL. In addition, GRHL2 knockout induced nearly complete loss of CTSL chromatin binding, demonstrating its role as a recruiter of CTSL to the chromatin. KDM4C serves as either a direct or indirect suppressor for CTSL-mediated H3 cleavage. The latter one was largely mediated by redox imbalance. Metabolomic profiling showed that KDM4C inhibition strongly dampened intracellular glutathione (GSH) levels and hence produced more reactive oxygen species (ROS) and alleviated mitochondrial respiration. This procedure was grounded on the mechanism that KDM4C blockade decreased expression of GSH synthesis rate-limiting enzyme glutamate-cysteine ligase catalytic subunit (GCLC) via CTSL-mediated H3 tail clipping. Knockout of CTSL rescued KDM4C blockade-associated metabolic dysfunction, confirming KDM4C links redox homeostasis and chromatin remodeling. Finally, we found KDM4C-CTSL-GSH axis was associated with cisplatin resistance in TNBC patients. Co-targeting KDM4C and GSH production efficiently improved cisplatin response in TNBC cells. Our study provided detailed and unbiased evidence supporting a novel non-canonical role of KDM4C in preventing CTSL-mediated histone clipping and maintaining redox balance, opening up possibilities to improve chemotherapy through targeting this circus. Citation Format: Zheqi Li, Guillermo Peluffo, Laura E. Stevens, Xintao Qiu, Shawn B. Egri, Malvina Papanastasiou, Natalie Kingston, Clive S. D'Santos, Eva Papachristou, Kyle Evans, Ji-Heui Seo, Kendell Clement, Daniel Temko, Muhammad Ekram, Anton Simeonov, Stephen C. Kales, Ganesha Rai, Madhu Lal-Nag, David J. Maloney, Ajit Jadhav, Franziska Michor, Alex Meissner, Jason S. Carroll, Matthew L. Freedman, Henry W. Long, Jacob D. Jaffe, Kornelia Polyak. KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A018.

Heparin-induced thrombocytopenia (HIT) is a rare, but deadly disease that occurs in a small percentage of patients following administration of heparin. In these patients, heparin interacts with platelet factor 4 (PF4) resulting in an immune response to the heparin/PF4 complex. This immune complex can then bind to the immune receptor on the platelet, FcγRIIa, to induce platelet activation, clot formation, thrombosis, consumption, and bleeding. This potentially deadly disease results in both a thrombotic event as well as bleeding. Currently, there is only one FDA-approved intervention for HIT, the direct thrombin inhibitor argatroban. However, many patients on argatroban remain at risk for thrombosis, bleeding, and in some cases death. Therefore, we sought to develop a new drug to intervene in patients with HIT by targeting the 12-lipoxygenase enzyme in the platelet which regulates FcγRIIa activity in the platelet. Using mouse models of HIT as well as ex vivo administration of the 12-lipoxygenase inhibitor VLX-1005 (previously known as ML355). In mice expressing the human immune receptor on their platelets, we were able to show that administration of VLX-1005 following induction of HIT in these mice resulted in a blunted thrombocytopenia as well as reduced platelet activation and thrombus formation in the blood. We further demonstrated that coagulation was not impacted by VLX-1005 using thromboelestography while argatroban significantly delayed onset of coagulation and clot formation. Furthermore, bleeding time in these mice was not altered with VLX-1005, while mice on argatroban required cauterization of their tails to stop the bleeding. Finally, Human whole blood was shown in whole blood aggregometry as well as high shear arterial flow chamber experiments to be protected from platelet activation and clot formation in the presence of VLX-1005. The studies presented here demonstrate the potential effectiveness of VLX-1005 in intervention of platelet activation, clot formation, and thrombosis in both mouse models and human blood and support VLX-1005 as a new class of drug for the treatment of HIT in patients without the risk of bleeding.

Type 1 diabetes (T1D) has become viewed as an autoimmune disease initiated and propagated by stress responses in islet β cells. The integrated stress response (ISR) is activated upon inflammatory signaling and delimits protein production through the actions of phospho-eIF2α. We hypothesized that inhibition of the β cell ISR will protect against β cell immunogenicity and development of T1D. To test this hypothesis, we studied the effect of ISR inhibition using ISRIB (p-eIF2α inhibitor) and VLX-10 (human 12-lipoxygenase inhibitor) on the response of isolated human islets and EndoC-βH1 cells to inflammatory cytokines (IFN-γ+IL-1β) and studied the effect of ISR inhibition on T1D outcomes in NOD mice. Human islets exposed to cytokines exhibited an increase in p-eIF2α and suppression of mRNA translation initiation (by polysome profiling) , consistent with activation of the ISR. Inhibition of the ISR with ISRIB or VLX-10promoted production of PD-L1, an immune checkpoint protein that suppresses the immune response, in human islets and EndoC-βH1 cells. This finding suggested that blockade of the ISR in β cells might thwart immune attack. To assess if inhibition of the ISR using the human-specific 12-lipoxygenase inhibitor VLX-10modifies T1D progression, we generated mice in which the gene encoding mouse 12-lipoxygenase gene was replaced by the corresponding human gene. These “humanized” mice were subsequently backcrossed onto the NOD background, then treated in the prediabetic phase with VLX-1005. Male and female humanized mice developed T1D at the expected frequency, but those receiving VLX-10showed significant delay of T1D onset. Consistent with the suppression of autoimmunity, insulitis was significantly reduced and β cell mass was significantly increased in treated animals. Our results support the targeting of the ISR through inhibition of the 12-lipoxygenase enzyme provides an opportunity for the modification of T1D disease progression. Disclosure F.Huang: None. A.Kulkarni: None. F.Syed: None. D.J.Maloney: Employee; Veralox Therapeutics. S.A.Tersey: n/a. R.G.Mirmira: None. Funding NIH (UDK127786-02)

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration–response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.