Second-generation Compounds Research Articles

Current Approaches and Novel Agents in the Treatment of Chronic Lymphocytic Leukemia.

The treatment of CLL has evolved from traditional chemoimmunotherapy (CIT) to an increasing number of targeted and biologic approaches. Randomized trials have demonstrated superiority of covalent bruton tyrosine kinase inhibitors (cBTKis) over CIT, and second-generation compounds such as acalabrutinib and zanubrutinib appear to have a more favorable efficacy/safety profile than ibrutinib. The noncovalent BTKi, pirtobrutinib, has shown impressive activity after failure of the cBTKis and is quite tolerable. The Bcl-2 inhibitor venetoclax plus a CD20, generally obinutuzumab, provides a high level of efficacy as initial treatment or after failure on a cBTKi, with many patients achieving a state of undetectable minimal residual disease. Promising novel approaches include BTK degraders, bispecific antibodies, and chimeric antigen receptor T-cell (CAR-T)-cell therapy. What is clear is that CIT is archaic, and current and future targeted approaches will continue to improve the outcome for patients with chronic lymphocytic leukemia.

Effectiveness of Two New Endochin-like Quinolones, ELQ-596 and ELQ-650, in Experimental Mouse Models of Human Babesiosis.

Endochin-like quinolones (ELQs) define a class of small molecule antimicrobials that target the mitochondrial electron transport chain of various human parasites by inhibiting their cytochrome bc1 complexes. The compounds have shown potent activity against a wide range of protozoan parasites, including the intraerythrocytic parasites Plasmodium and Babesia, the agents of human malaria and babesiosis, respectively. First-generation ELQ compounds were previously found to reduce infection by Babesia microti and Babesia duncani in animal models of human babesiosis but achieved a radical cure only in combination with atovaquone and required further optimization to address pharmacological limitations. Here, we report the identification of two second-generation 3-biaryl ELQ compounds, ELQ-596 and ELQ-650, with potent antibabesial activity in vitro and favorable pharmacological properties. In particular, ELQ-598, a prodrug of ELQ-596, demonstrated high efficacy as an orally administered monotherapy at 10 mg/kg. The compound achieved radical cure in both the chronic model of B. microti-induced babesiosis in immunocompromised mice and the lethal infection model induced by B. duncani in immunocompetent mice. Given its high potency, favorable physicochemical properties, and low toxicity profile, ELQ-596 represents a promising drug for the treatment of human babesiosis.

Combating PDAC Drug Resistance: The Role of Ref-1 Inhibitors in Accelerating Progress in Pancreatic Cancer Research.

Pancreatic Ductal Adenocarcinoma (PDAC) remains one of the most lethal solid tumor diagnoses given its limited treatment options and dismal prognosis. Its complex tumor microenvironment (TME), heterogeneity, and high propensity for drug resistance are major obstacles in developing effective therapies. Here, we highlight the critical role of Redox effector 1 (Ref-1) in PDAC progression and drug resistance, focusing on its redox regulation of key transcription factors (TFs) such as STAT3, HIF1α, and NF-κB, which are pivotal for tumor survival, proliferation, and immune evasion. We discuss the development of novel Ref-1 inhibitors, including second-generation compounds with enhanced potency and improved pharmacokinetic profiles, which have shown significant promise in preclinical models. These inhibitors disrupt Ref-1's redox function, leading to decreased TF activity and increased chemosensitivity in PDAC cells. We further detail our utilization of advanced preclinical models, such as 3D spheroids, organoids, and Tumor-Microenvironment-on-Chip (T-MOC) systems, which better simulate the complex conditions of the PDAC TME and improve the predictive power of therapeutic responses. By targeting Ref-1 and its associated pathways, in conjunction with improved models, more replicative of PDAC's TME, we are focused on approaches which hold the potential to overcome current therapeutic limitations and advance the development of more effective treatments for PDAC. Our findings suggest that integrating Ref-1 inhibitors into combination therapies could disrupt multiple survival mechanisms within the tumor, offering new hope for improving outcomes in this challenging cancer.

Potential exposure of native wildlife to anticoagulant rodenticides in Gran Canaria (Canary Islands, Spain): Evidence from residue analysis of the invasive California Kingsnake (Lampropeltis californiae)

Anticoagulant rodenticides (ARs), particularly second-generation compounds (SGARs), are extensively used in pest management, impacting non-target wildlife. The California kingsnake (Lampropeltis californiae), an invasive species in Gran Canaria, is under a control plan involving capture and euthanasia. This research aimed to detect 10 different ARs in these snakes, explore geographical and biometrical factors influencing AR exposure, and assess their potential as sentinel species for raptors, sharing similar foraging habits. Liver samples from 360 snakes, euthanized between 2021 and 2022, were analysed for ARs using LC-MS/MS. Results showed all detected rodenticides were SGARs, except for one instance of diphacinone. Remarkably, 90 % of the snakes tested positive for ARs, with over half exposed to multiple compounds. Brodifacoum was predominant, found in over 90 % of AR-positive snakes, while bromadiolone and difenacoum were also frequently detected but at lower levels. The study revealed that larger snakes and those in certain geographic areas had higher AR concentrations. Snakes in less central or more peripheral areas showed lower levels of these compounds. This suggests a correlation between the snakes' size and distribution with the concentration of ARs in their bodies. The findings indicate that the types and prevalence of ARs in California kingsnakes on Gran Canaria mirror those in the island's raptors. This similarity suggests that the kingsnake could serve as a potential sentinel species for monitoring ARs in the ecosystem. However, further research is necessary to confirm their effectiveness in this role.

DIPG-17. TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA USING ALLELE-SPECIFIC SIRNA TARGETING H3.3 K27M MUTANT HISTONE

Abstract In recent years CNS tumors have surpassed leukemia and lymphoma as the deadliest forms of pediatric cancer, yet many of these tumors remain resistant to treatment. Understanding of the molecular pathology of a particularly fatal pediatric glioma, diffuse intrinsic pontine glioma (DIPG), has improved dramatically over the past decade. However, this knowledge has not yet yielded new targeted therapeutics or advances in treatment. The majority of DIPG tumors (~70-80%) harbor a K27M mutation in the histone H3.3 gene resulting in significant epigenetic changes which drive tumorigenesis. We are utilizing cutting edge siRNA chemistries to develop a therapeutic specifically targeting this oncohistone H3.3 K27M. It has been shown by others that ex vivo disruption of the H3.3 K27M mutant significantly delays tumor development in orthotopic xenograft models and could be a promising target for treatment of disease. New siRNA chemistries allow broad distribution and silencing throughout the brain with durable silencing for months. Utilizing this technology, we have engineered siRNA compounds that can discriminate between wild-type (WT) and K27M mutant H3.3 alleles. Our first-generation compounds achieved >95% silencing of K27M mutant with only 40% silencing of WT H3.3. We have developed second-generation compounds to improve the discrimination between the two alleles. These compounds allow us to selectively knockdown the mutant driver of DIPG tumorigenesis and we hypothesize that silencing of this oncohistone will revert the epigenetic landscape of DIPG to a more wild-type form detrimentally impacting tumor growth and progression. Together the in vivo potency of the new siRNA chemistries combined with the precision allele-specific targeting of mutant K27M oncohistone may be a promising path towards development of an effective and safe therapy for DIPG.

Schizophrenia Synaptic Pathology and Antipsychotic Treatment in the Framework of Oxidative and Mitochondrial Dysfunction: Translational Highlights for the Clinics and Treatment.

Schizophrenia is a worldwide mental illness characterized by alterations at dopaminergic and glutamatergic synapses resulting in global dysconnectivity within and between brain networks. Impairments in inflammatory processes, mitochondrial functions, energy expenditure, and oxidative stress have been extensively associated with schizophrenia pathophysiology. Antipsychotics, the mainstay of schizophrenia pharmacological treatment and all sharing the common feature of dopamine D2 receptor occupancy, may affect antioxidant pathways as well as mitochondrial protein levels and gene expression. Here, we systematically reviewed the available evidence on antioxidants' mechanisms in antipsychotic action and the impact of first- and second-generation compounds on mitochondrial functions and oxidative stress. We further focused on clinical trials addressing the efficacy and tolerability of antioxidants as an augmentation strategy of antipsychotic treatment. EMBASE, Scopus, and Medline/PubMed databases were interrogated. The selection process was conducted in respect of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria. Several mitochondrial proteins involved in cell viability, energy metabolism, and regulation of oxidative systems were reported to be significantly modified by antipsychotic treatment with differences between first- and second-generation drugs. Finally, antioxidants may affect cognitive and psychotic symptoms in patients with schizophrenia, and although the evidence is only preliminary, the results indicate that further studies are warranted.

Abstract 3419: TY-1091, a highly selective and potent second-generation RET inhibitor, demonstrates superior antitumor activity in multiple RET-mutant models

Abstract Proto-oncoprotein RET (rearranged during transfection) is a receptor tyrosine kinase and belongs to the cadherin superfamily. RET fusion proteins and gatekeeper mutations represent strong cancer drivers involved in the development of a variety of cancers. Although selective RET inhibitors selpercatinib (LOXO-292) and pralsetinib (BLU667) were recently marketed for patients with RET-dependent NSCLC and thyroid cancers, RET solvent front mutations G810R/S/C/V and other secondary mutations such as Y806C/N and V728A have been identified as mechanisms counting for acquired resistance to the two drugs and limits the applications of the kinase inhibitors. To provide novel RET inhibitors effective to a broader spectrum of RET fusions and mutations, we identified TY-1091 as a novel next-generation RET inhibitor through the in-house RET program and systemic screening of RET compound candidates. TY-1091 was characterized for its anti-tumor activity through in vitro and in vivo testing of a variety of RET-dependent tumor models including a panel of 17 engineered RET mutant Ba/F3 cell lines and 2 cancer cell models. The results show that TY-1091 inhibits wild type and major RET mutants (IC50, nM): RET G810S (9.5 nM), RET V804M/L/E (2.8-12.6 nM), and double mutants RET V804M/G810S (23.5 nM) and M918T/G810S (47.0 nM) through a biochemical screening against a panel of 17 engineered RET mutant Ba/F3 cell lines. Importantly, the inhibitory activity of TY-1091 is much higher than that of first-generation RET inhibitor Cabozantinib, and comparable to other second-generation compounds LOXO-292 and BLU-667. Consistent with its mode of action, TY-1091 grants extraordinary inhibition effects to tumor cell proliferation compared to its peer compounds LOXO-292 and BLU-667 (including TT (thyroid cancer, RET C634W) and LC2/ad (NSCLC, CCDC6-RET)), and the inhibition of the RET pathway activation, i.e., inhibition of RET phosphorylation (IC50 < 1 nM), SHC phosphorylation (IC50 = 4.6 nM) and ERK phosphorylation (IC50 = 9.8 nM) in Ba/F3-KIF5B-RET cells further validated the above in vitro phenotype. The therapeutic potential of TY-1091 was then further validated through mouse pharmacology in that TY-1091 demonstrated remarkable anti-tumor efficacy in a variety of xenograft models including Ba/F3 KIF5B-RET (wt), Ba/F3 KIF5B-RET (V804L), TT (thyroid cancer, RET C634W), and LC2/ad (NSCLC, CCDC6-RET). Detailed data will be presented. In summary, TY-1091 is a highly potent, orally available, and safe small molecule inhibitor to pan-RET mutations in cancer, and may attenuate SFMs-mediated resistance to existing RET therapy. The IND clearance from the US FDA was received and Phase I clinical investigations of TY-1091 shall be launched in the US soon. *To Whom Correspondence should be addressed to: Jun Li, Chengshan Niu and Yusheng Wu Citation Format: Chengshan Niu, Maolin Zheng, Huan Wang, Kaige Ji, Meihua Li, Guohui Wang, Rongzhen Ni, Apeng Liang, Aishen Gong, Yazhen Zhang, Hui Su, Mingyu Jiang, Shaoqing Chen, Xiugui Chen, Jun Li, Yusheng Wu. TY-1091, a highly selective and potent second-generation RET inhibitor, demonstrates superior antitumor activity in multiple RET-mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3419.

Conformation-dependent ligand hot spots in the spliceosomal RNA helicase BRR2.

The conversion of hits to leads in drug discovery involves the elaboration of chemical core structures to increase their potency. In fragment-based drug discovery, low-molecular-weight compounds are tested for protein binding and are subsequently modified, with the tacit assumption that the binding mode of the original hit will be conserved among the derivatives. However, deviations from binding mode conservation are rather frequently observed, but potential causes of these alterations remain incompletely understood. Here, two crystal forms of the spliceosomal RNA helicase BRR2 were employed as a test case to explore the consequences of conformational changes in the target protein on the binding behaviour of fragment derivatives. The initial fragment, sulfaguanidine, bound at the interface between the two helicase cassettes of BRR2 in one crystal form. Second-generation compounds devised by structure-guided docking were probed for their binding to BRR2 in a second crystal form, in which the original fragment-binding site was altered due to a conformational change. While some of the second-generation compounds retained binding to parts of the original site, others changed to different binding pockets of the protein. A structural bioinformatics analysis revealed that the fragment-binding sites correspond to predicted binding hot spots, which strongly depend on the protein conformation. This case study offers an example of extensive binding-mode changes during hit derivatization, which are likely to occur as a consequence of multiple binding hot spots, some of which are sensitive to the flexibility of the protein.

Tau positron emission tomography in tauopathies: A narrative review

Aggregation of misfolded tau in the brain is a major pathological feature common in various neurodegenerative disorders known as tauopathies, including Alzheimer’s disease, progressive supranuclear palsy, corticobasal syndrome, and dementia with Lewy bodies. Tauopathies are collection of diseases with varied overlapping symptoms and complicated manifestations. Consequently, it is crucial to be able to assess tau deposits <i>in vivo</i>. Over the past decade, tau-specific radioligands for positron emission tomography (PET) have been developed and tested, including first-generation compounds (e.g., <sup>18</sup>F-THK5317, <sup>18</sup>F-THK5351, <sup>18</sup>F-AV1451, and <sup>11</sup>C-PBB3) and second-generation compounds (<sup>18</sup>F-MK-6240, <sup>18</sup>F-RO-948, and <sup>18</sup>F-PI-2620). With the recent advances of tau PET tracers, assessing the pattern of tau deposition <i>in vivo</i> is possible. These methods will allow accurate diagnosis of tauopathies and monitoring of disease progression. In this mini review, we summarize current findings from studies using tau PET tracers regarding neuropathological characteristics, clinical implications, and potential applications of tau PET. We also discuss methodological considerations for appropriate use of these technologies and discuss what has been learned from these findings.

Interest of the faecal and plasma matrix for monitoring the exposure of wildlife or domestic animals to anticoagulant rodenticides

Anticoagulant rodenticides (ARs), particularly second-generation compounds (SGAR), are known to be a potential threat to unintended species due to their tissue persistence. The liver is the storage tissue of ARs and is a matrix of choice in diagnosing exposure and intoxication of non-target fauna. However, it is only available on dead animals. Blood and faeces can be used on living animals. These two biological matrices were compared in terms of their relevance to exposure to ARs. In addressing this question, we compared the faecal, plasma and liver concentrations of bromadiolone, one of the SGAR frequently implicated in wildlife exposure. We studied this comparison at the individual level and at the population level, considering three influencing factors: dose, sex and time. Our findings demonstrate that faecal analyses are more valuable than plasma analyses for monitoring AR exposure of domestic and wild animals, even if faecal concentrations cannot be correlated with liver concentrations.

EDP-938, a new antiviral with inhibitory activity against the nucleoprotein of the respiratory syncytial virus

The absence of an effective vaccine against respiratory syncytial virus (RSV) has led to the development of various drugs with the ability to inhibit or block its replicative activity. The first generation, called fusion inhibitors, bind to the protein on the viral surface and prevent the virus from binding and entering the cell. However, its low efficacy has determined the start of studies with second-generation compounds capable of binding or blocking the nucleoprotein (N); most of these compounds are analogs of 1,4-benzodiazepines. EDP-938 has shown high efficacy against RSV. The first trials in humans have shown that this antiviral is rapidly absorbed after oral administration and has a half-life of between 11-18 hours Administration for seven days of multiple oral doses of up to 600 mg/day or 300 mg/day/twice a day, there were hardly any significant adverse effects and the viral load in the lower respiratory tract decreased significantly.

Long-term effects of neonicotinoids on reproduction and offspring development in the copepod Acartia tonsa

Neonicotinoids (NEOs) are neurotoxic pesticides acting as nicotinic acetylcholine receptor agonists. NEOs' efficacy against pest insects has favoured their spreading use in agriculture, but their proven effectiveness against non-target insects in terrestrial and aquatic ecosystems also raised concern over their environmental impact. Crustaceans were often studied for the impacts of NEOs due to their economic values and nervous' system similarity with insects. However, most studies on crustaceans focused on acute effects or exposure of early-life stages, while long-term effects were seldom explored. The present study aimed to assess the potential long-term effects of four commercially available NEOs on the reproduction and offspring of the calanoid copepod Acartia tonsa, a key species in the food webs of several coastal and estuarine environments. NEOs were confirmed as potent interferents of copepod reproduction. The first-generation compound acetamiprid significantly inhibited egg production and hatching ratio at 10 ng L−1, while larval survival and development were affected at 81 ng L−1. Similarly, the first-generation compound thiacloprid significantly inhibited the hatching ratio and larval development at 9 ng L−1, while it did not affect egg production and larval survival. Second-generation compounds were less toxic than acetamiprid and thiacloprid: clothianidin affected significantly only larval development of the offspring at 62 ng L−1, while thiamethoxam was not toxic at both the tested concentrations (8 ng L−1 and 84 ng L−1). These data evidenced that effects on copepods may occur at concentrations below the chronic aquatic life benchmarks reported by USEPA for acetamiprid (2100 ng L−1) and thiacloprid (970 ng L−1), suggesting that long-term effects of NEOs have been underestimated. A comparison with environmental concentrations evidenced that NEO-mediated effects on copepods are more liable in coastal areas receiving discharge from wastewater treatment plants or diffuse inputs from agricultural land during pesticide application periods.

Anticoagulant rodenticide blood-clotting dose-responses and resistance factors for Tyrosine139Cysteine (Y139C) heterozygous- and homozygous-resistant house mice (Mus musculus).

The house mouse (Mus musculus) is a globally distributed rodent pest species against which anticoagulant rodenticides are widely used for the protection of human and animal health and the conservation of threatened wildlife. Anticoagulant-resistant house mice have been known for more than half a century. A house mouse strain was developed in the laboratory that was homozygous resistant for the single nucleotide polymorphism (SNP) Tyrosine139Cysteine (Y139C) and, subsequently, heterozygous resistant animals were produced from this strain by crossing with the homozygous susceptible strain. Using blood clotting response tests, resistance factors at the ED50 level in the homozygous resistant strain for the first-generation anticoagulants warfarin, chlorophacinone, diphacinone and coumatetralyl were in the range 31.5 to 628.0 for males (M) and 21.6 to 628.0 for females (F), thus indicating that Y139C house mice are substantially resistant to all these substances. Resistance factors at the ED50 level for the homozygous strain generated against the second-generation compounds were: brodifacoum (M, 1.7; F, 1.9), bromadiolone (M, 16.6; F, 21.0), difenacoum (M, 1.2; F, 2.7), difethialone (M, 1.5; F, 1.5), and flocoumafen (M, 0.9; F, 1.2). Equivalent values for the heterozygous strain were: brodifacoum (M, 1.6; F, 1.4), bromadiolone (M, 5.6; F, 6.5), difenacoum (M, 1.0; F, 1.3), difethialone (M, 1.1; F, 1.1), flocoumafen (M, 0.9; F, 1.1). Y139C SNP homozygous resistant mice are more resistant to anticoagulants than heterozygous resistant animals. All first-generation anticoagulants are highly resisted and, among the second-generation compounds, Y139C mice are resistant to bromadiolone and sometimes to difenacoum. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Anticoagulant Rodenticide Contamination of Terrestrial Birds of Prey from Western Canada: Patterns and Trends, 1988-2018.

As the dominant means for control of pest rodent populations globally, anticoagulant rodenticides (ARs), particularly the second‐generation compounds (SGARs), have widely contaminated nontarget organisms. We present data on hepatic residues of ARs in 741 raptorial birds found dead or brought into rehabilitation centers in British Columbia, Canada, over a 30‐year period from 1988 to 2018. Exposure varied by species, by proximity to residential areas, and over time, with at least one SGAR residue detected in 74% of individuals and multiple residues in 50% of individuals. By comparison, we detected first‐generation compounds in <5% of the raptors. Highest rates of exposure were in barred owls (Strix varia), 96%, and great horned owls (Bubo virginianus), 81%, species with diverse diets, including rats (Rattus norvegicus and Rattus rattus), and inhabiting suburban and intensive agricultural habitats. Barn owls (Tyto alba), mainly a vole (Microtus) eater, had a lower incidence of exposure of 65%. Putatively, bird‐eating raptors also had a relatively high incidence of exposure, with 75% of Cooper's hawks (Accipiter cooperii) and 60% of sharp‐shinned hawks (Accipiter striatus) exposed. Concentrations of SGARs varied greatly, for example, in barred owls, the geometric mean ∑SGAR = 0.13, ranging from <0.005 to 1.81 μg/g wet weight (n = 208). Barred owls had significantly higher ∑SGAR concentrations than all other species, driven by significantly higher bromadiolone concentrations, which was predicted by the proportion of residential land within their home ranges. Preliminary indications that risk mitigation measures implemented in 2013 are having an influence on exposure include a decrease in mean concentrations of brodifacoum and difethialone in barred and great horned owls and an increase in bromodialone around that inflection point. Environ Toxicol Chem 2022;41:1903–1917. © 2022 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

TXH11106: A Third-Generation MreB Inhibitor with Enhanced Activity against a Broad Range of Gram-Negative Bacterial Pathogens.

The emergence of multi-drug-resistant Gram-negative pathogens highlights an urgent clinical need to explore and develop new antibiotics with novel antibacterial targets. MreB is a promising antibacterial target that functions as an essential elongasome protein in most Gram-negative bacterial rods. Here, we describe a third-generation MreB inhibitor (TXH11106) with enhanced bactericidal activity versus the Gram-negative pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa compared to the first- and second-generation compounds A22 and CBR-4830, respectively. Large inocula of these four pathogens are associated with a low frequency of resistance (FOR) to TXH11106. The enhanced bactericidal activity of TXH11106 relative to A22 and CBR-4830 correlates with a correspondingly enhanced capacity to inhibit E. coli MreB ATPase activity via a noncompetitive mechanism. Morphological changes induced by TXH11106 in E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa provide further evidence supporting MreB as the bactericidal target of the compound. Taken together, our results highlight the potential of TXH11106 as an MreB inhibitor with activity against a broad spectrum of Gram-negative bacterial pathogens of acute clinical importance.

Abstract 1452: Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models

Abstract Targeted therapy for cancer using small molecules has progressed exponentially, but agents that can affect cancer cells rather than non-tumorigenic cells are crucial to avoid pernicious side effects. AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein with DNA repair activity and redox signaling activity as major functions. The DNA repair function is indispensable for cell survival. Its role as a redox factor that stimulates the DNA binding activity of numerous transcription factors (TFs) such as HIF-1α, NFΚB, STAT3, and AP-1 tends to be dysregulated in cancer cells. Ref-1 is overexpressed in many cancers including, aggressive and invasive pancreatic ductal adenocarcinoma (PDAC), which is the 3rd leading cause of death in U.S. The dense stroma of PDAC primarily constituting cancer-associated fibroblasts (CAFs) makes it highly hypoxic, nutrient poor, and drug resistant. Current treatment regimens offer only modest improvement in survival. Targeting the PDAC stroma or ECM did not result in clinical benefit thus far presumably due to complex interactions between PDAC and its TME. Better treatments options that selectively target the tumor within its protective stroma are hence needed. Our group was able to generate a Ref-1 redox inhibitor, APX3330, that completed Phase I clinical trial (NCT03375086) with a good safety profile, verified target engagement and a recommended phase II dose. However, its potency in preclinical models is in the high µM range indicating the need for more potent second-generation inhibitors. Based on initial SAR studies, we selected 13 second-generation compounds from &amp;gt; 350 that were further characterized for positive properties including increased efficacy for cell killing, mouse and human S9 fraction metabolic stability, plasma half-life, and in silico ADMET properties. Target engagement studies involving blockade of TF activity via luciferase assay and EMSA as well as validation of direct interaction of these inhibitors with Ref-1 using thermal shift assay are ongoing. In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly while sparing the CAFs. These findings were confirmed in vivo with xenografts co-implanted with tumor and CAF lines. Gene expression and mitochondria functional data revealed Ref-1's control of TCA cycle in tumor cells, but not in CAFs. To confirm and compare the effects of Ref-1 redox signaling inhibitors in cells, CRISPR editing was used to generate Ref-1 redox deficient cell lines (Ref-1C65A). As confirmation of significantly reduced Ref-1 redox activity, PDAC cells expressing Ref-1C65A did not induce hypoxia marker (CA9) under hypoxia, similar to when Ref-1 was knocked down or blocked via small molecule inhibitor. Ref-1 redox signaling and validation for selective killing of PDAC cells leaving the stomal cells undisturbed paves the way to improved PDAC treatment. Citation Format: Silpa Gampala, Nayela Chowdhury, Olivia Babb, Rachel A. Caston, Randall S. Wireman, Hye-ran Moon, George Sandusky, Emily Hulsey, Bumsoo Han, Millie M. Georgiadis, Sara K. Quinney, Andi R. Masters, James H. Wikel, Mark R. Kelley, Melissa L. Fishel. Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1452.

Amisulpride - is it as all other medicines or is it different? An update.

Amisulpride is an antipsychotic available in Europe since 1990s, in Poland since 2000. Subsequent years brought to Polish market more second-generation compounds such as ziprasidone and aripiprazole. In 2018, the Agency for Health Technology Assessment and Tariff System issued positive recommendation for lurasidone in schizophrenia (Recommendation 30/2018) facilitating its entry to the market. Thanks to new molecules, therapeutic possibilities of medicines consequently rise, however, higher number of available substances of different properties brings also more dilemmas which one to pick. Since new publications of comparative drug trials, meta-analyses and systematic reviews are issued regularly, the authors present herein publications issued within last ten years focusing on amisulpride as opposed to other neuroleptics used in Poland. Although in many aspects it is equivalent to other atypical antipsychotics, it still has some advantages. Amisulpride seems to have better outcome than classic and atypical neuroleptics when it comes to depressive symptoms and predominant negative symptoms. It might also be superior to haloperidol in inducing symptomatic remission in first episode schizophrenia. Except for prolactin increase its side effects profile is favorable - it rarely leads to extrapyramidal symptoms (which are dose-dependent) and sedation. Therefore many patients accept treatment with amisulpride for its measurable clinical gains, such as improvement of positive symptoms and higher quality of life, compared to typical neuroleptics. Pharmacokinetics of amisulpride also encourage its wider use, especially when there is either a need for combined psychopharmacotherapy or comorbidity with general medical condition rises a need for somatic parallel treatment.

Development of Polyamine-Substituted Triphenylamine Ligands with High Affinity and Selectivity for G-Quadruplex DNA.

Currently, significant efforts are devoted to designing small molecules able to bind selectively to guanine quadruplexes (G4s). These noncanonical DNA structures are implicated in various important biological processes and have been identified as potential targets for drug development. Previously, a series of triphenylamine (TPA)-based compounds, including macrocyclic polyamines, that displayed high affinity towards G4 DNA were reported. Following this initial work, herein a series of second-generation compounds, in which the central TPA has been functionalised with flexible and adaptive linear polyamines, are presented with the aim of maximising the selectivity towards G4 DNA. The acid-base properties of the new derivatives have been studied by means of potentiometric titrations, UV/Vis and fluorescence emission spectroscopy. The interaction with G4s and duplex DNA has been explored by using FRET melting assays, fluorescence spectroscopy and circular dichroism. Compared with previous TPA derivatives with macrocyclic substituents, the new ligands reported herein retain the G4 affinity, but display two orders of magnitude higher selectivity for G4 versus duplex DNA; this is most likely due to the ability of the linear substituents to embrace the G4 structure.

Secreted Factors Determine Resistance to Idelalisib in Marginal Zone Lymphoma Models of Resistance

Background . PI3Kδ is expressed in B-cells and has a central role in the B-cell receptor signaling in B-cell derived malignancies. Idelalisib was the first-in-class PI3Kδ inhibitors and several second-generation compounds are undergoing clinical investigation as single agents and in combinations. To identify modalities to overcome the resistance that develops to this class of agents, we have developed two idelalisib-resistant models derived from splenic marginal zone lymphoma (SMZL) cell lines. Materials and Methods. Cells were kept under idelalisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome and miRNA profiling by RNA-Seq, whole exome sequencing (WES), lipidomics profiling, pharmacological screening (348 compounds), and FACS analysis. Cytokines and growth factor secretion was performed by ELISA. Results. Two RES models were obtained from VL51 and Karpas1718 with 7-10 fold times higher IC50s than PAR counterparts. In both models, conditioned media from RES cells transferred the resistance in the PAR cells. While WES did not identify somatic mutations associated with resistance, RNA-Seq and lipidomics analyses showed that the two cell lines had developed resistance activating different modalities. The VL51 RES model showed an enrichment in BCR-TLR-NFkB (TLR4, CD19, SYK), IL6-STAT3 (IL6, CD44), chemokines (CXCL10, CXCR4, CXCR3) and PDGFR (PDGFRA, PRKCE) signatures, paired with increased p-AKT and p-BTK levels, decreased cardiolipins and sphingomyelins levels, and increased levels of specific triacylglycerols and glycerophosphocholines. In particular, there was an over-expression of surface expression of PDGFRA and secretion of IL6 in the medium. Silencing of both IL6and PDGFRA by siRNAs reverted the resistance, while the silencing of the individual genes had only a partial effect. These data were paired with the acquired sensitivity to the PDGFR inhibitor masitinib, identified in the pharmacologic screening. In the Karpas1718 model, we observed an increased p-AKT activity with an enrichment for B-cell activation signatures (RAG1, RAG2, TCL1A), proliferation (E2F2, MKI67), ERBB signaling (HBEGF, NRG2, ERRB4), increased levels of some triacylglycerols and repressed levels for specific glycerophosphocholines. HBEGF secretion was confirmed by ELISA. The addition of recombinant HBEGF to the medium induced resistance in the PAR cells. Combination with the pan ERBB inhibitor lapatinib was beneficial in the K1718 RES. Recombinant HBEGF also induced resistance to the BTK inhibitor ibrutinib in the PAR cells and in the mantle cell lymphoma SP-53 cell line. Specific members of the let-7 family of miRNAs were repressed in the RES lines derived from both cell lines, indicating the involvement of miRNA deregulation in the mechanism of resistance. Indeed, let-7 members are known to directly target IL6-STAT3 and cytokine signaling cascade, as well PI3K-AKT network. In solid tumors, let-7 members are also expressed at low levels in tumors with constitutive active ERBB signaling, in accordance with the activation of ERBB pathway and p-AKT we observed in our Karpas1718model. Experiments with a LIN28B inhibitor are now on-going. Finally, we validated the findings across a panel of 34 B-cell lymphoma cell lines, in which IL6, PDGFRA, HBEGF and LIN28 expression levels were negatively correlated with idelalisib sensitivity, while the latter was positively correlated with let-7 levels (P &lt;0.05). Conclusions. We developed two distinct models derived from MZL of secondary resistance to the PI3Kδ inhibitor idelalisib. We identified treatments that might overcome resistance to idelalisib and are worth of further investigations. The two models, driven by different biologic processes, will allow the evaluation of further alternative therapeutic approaches. Disclosures Stathis: PharmaMar: Other: Renumeration; ADC Therapeutics: Other: Institutional research funding; Abbvie: Other: Renumeration; Bayer: Other: Institutional research funding; Novartis: Other: Institutional research funding; MEI-Pharma: Other: Institutional research funding; Roche: Other: Institutional research funding; Pfizer: Other: Institutional research funding; Merck: Other: Institutional research funding. Stuessi:Gilead: Speakers Bureau. Zucca:Gilead: Honoraria, Other: travel grant. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; Jazz Pharmaceuticals: Other: travel grants; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants.

Enantiomer of the novel flexible heteroarotinoid, SL-1-09, blocks cell cycle progression in breast cancer cells

Flexible heteroarotinoids (Flex-Hets) are compounds with promising anti-cancer activities. SHetA2, a first-generation Flex-Het, has been shown to inhibit the growth of cervical, head and neck, kidney, lung, ovarian, prostate, and breast cancers. However, SHetA2's high lipophilicity, limited selectivity, low oral bioavailability, and complicated synthesis has led to the development of second-generation compounds, such as 1-(1-(naphthalen-1-yl)ethyl)-3-(4-nitrophenyl) thiourea or SL-1-09. Results from our lab show that SL-1-09 exhibits anti-cancer activities against ERα+ and ERα- breast cancer cells at micromolar concentrations. SL-1-09 is a mixture of two enantiomers, R and S. The objective of this study was to further analyze these enantiomers to determine their individual anti-cancer activities. Cell cycle analysis demonstrated that the percentage of cells in S-phase is reduced significantly when breast cancer cell lines MCF-7, T47D and MDA-MB-453 cells are treated with 5.0 μM of the S enantiomer. Consistent with this finding, treatment of these cells with the S enantiomer resulted in lower expression levels of cell cycle proteins. Overall, our data indicate that the S enantiomer shows greater growth inhibitory effects than the R form against ERα+ (MCF7 and T47D) and ERα- (MDA-MB-453) breast cancer cells, suggesting that the activity observed in SL-1-09 is most likely due to the ability of the S enantiomer to block cell cycle progression.