BACKGROUND: Delta-like ligand 3 (DLL3) is highly expressed on the cell surface of small cell lung cancer (SCLC), one of the most lethal malignancies, but minimally or not in normal tissues, making it an attractive target for SCLC. However, none of the DLL3-targeting antibody-drug conjugates (ADCs) have been approved for SCLC therapy yet. We developed DB-1314, the new anti-DLL3 ADC composed of a novel humanized anti-DLL3 monoclonal antibody (DB131401) conjugated with eight molecules of P1021 (topoisomerase I inhibitor), and described its preclinical profiles.
METHODS: The binding epitope for DB131401 and Rovalpituzumab was tested by biolayer interferometry. The binding affinity and specificity of DB-1314 to DLL3 and other homologous proteins were respectively measured by surface plasmon resonance and enzyme-linked immunosorbent assay. Internalization, bystander effects, and antibody-dependent cell-mediated cytotoxicity (ADCC) were assessed by respective assay. DLL3 was quantified by antibodies bound per cell assay and immunohistochemistry. In vitro and in vivo growth inhibition studies were evaluated in SCLC cell lines, and cell line/patient-derived xenograft models. The safety profile was measured in cynomolgus monkeys.
RESULTS: DB-1314 induces potent, durable, and dose-dependent antitumor effects in cells in vitro and in cell/patient-derived xenograft models in vivo. The killing activity of DB-1314 mechanically arises from P1021-induced DNA damage, whereby P1021 is delivered and released within tumor cells through DLL3-specific binding and efficient internalization. Bystander effects and ADCC also contribute to the antitumor activity of DB-1314. DB-1314 displays favorable pharmacokinetic and toxicokinetic profiles in rats and cynomolgus monkeys; besides, DB-1314 is well-tolerated at a dose of up to 60 mg/kg in monkeys.
CONCLUSIONS: These results suggest that DB-1314 may be a candidate ADC targeting DLL3 for the treatment of DLL3-positive SCLC, supporting further evaluation in the clinical setting.

OBJECTIVE: PARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1.
METHODS: A panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM).
RESULTS: Rucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib.
CONCLUSIONS: Our data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence \"induced synthetic lethality\" with PARPi.

BACKGROUND: Delta-like ligand 3 (DLL3) is aberrantly expressed on the surface of small-cell lung cancer (SCLC) and neuroendocrine prostate cancer cells. We assessed the safety and feasibility of the DLL3-targeted imaging tracer [89Zr]Zr-DFO-SC16.56 (composed of the anti-DLL3 antibody SC16.56 conjugated to p-SCN-Bn-deferoxamine [DFO] serving as a chelator for zirconium-89) in patients with neuroendocrine-derived cancer.
METHODS: We conducted an open-label, first-in-human study of immunoPET-CT imaging with [89Zr]Zr-DFO-SC16.56. The study was done at Memorial Sloan Kettering Cancer Center, New York, NY, USA. Patients aged 18 years or older with a histologically verified neuroendocrine-derived malignancy and an Eastern Cooperative Oncology Group performance status of 0-2 were eligible. An initial cohort of patients with SCLC (cohort 1) received 37-74 MBq [89Zr]Zr-DFO-SC16.56 as a single intravenous infusion at a total mass dose of 2·5 mg and had serial PET-CT scans at 1 h, day 1, day 3, and day 7 post-injection. The primary outcomes of phase 1 of the study (cohort 1) were to estimate terminal clearance half-time, determine whole organ time-integrated activity coefficients, and assess the safety of [89Zr]Zr-DFO-SC16.56. An expansion cohort of additional patients (with SCLC, neuroendocrine prostate cancer, atypical carcinoid tumours, and non-small-cell lung cancer; cohort 2) received a single infusion of [89Zr]Zr-DFO-SC16.56 at the same activity and mass dose as in the initial cohort followed by a single PET-CT scan 3-6 days later. Retrospectively collected tumour biopsy samples were assessed for DLL3 by immunohistochemistry. The primary outcome of phase 2 of the study in cohort 2 was to determine the potential association between tumour uptake of the tracer and intratumoural DLL3 protein expression, as determined by immunohistochemistry. This study is ongoing and is registered with ClinicalTrials.gov, NCT04199741.
RESULTS: Between Feb 11, 2020, and Jan 30, 2023, 12 (67%) men and six (33%) women were enrolled, with a median age of 64 years (range 23-81). Cohort 1 included three patients and cohort 2 included 15 additional patients. Imaging of the three patients with SCLC in cohort 1 showed strong tumour-specific uptake of [89Zr]Zr-DFO-SC16.56 at day 3 and day 7 post-injection. Serum clearance was biphasic with an estimated terminal clearance half-time of 119 h (SD 31). The highest mean absorbed dose was observed in the liver (1·83 mGy/MBq [SD 0·36]), and the mean effective dose was 0·49 mSv/MBq (SD 0·10). In cohort 2, a single immunoPET-CT scan on day 3-6 post-administration could delineate DLL3-avid tumours in 12 (80%) of 15 patients. Tumoural uptake varied between and within patients, and across anatomical sites, with a wide range in maximum standardised uptake value (from 3·3 to 66·7). Tumour uptake by [89Zr]Zr-DFO-SC16.56 was congruent with DLL3 immunohistochemistry in 15 (94%) of 16 patients with evaluable tissue. Two patients with non-avid DLL3 SCLC and neuroendocrine prostate cancer by PET scan showed the lowest DLL3 expression by tumour immunohistochemistry. One (6%) of 18 patients had a grade 1 allergic reaction; no grade 2 or worse adverse events were noted in either cohort.
CONCLUSIONS: DLL3 PET-CT imaging of patients with neuroendocrine cancers is safe and feasible. These results show the potential utility of [89Zr]Zr-DFO-SC16.56 for non-invasive in-vivo detection of DLL3-expressing malignancies.
BACKGROUND: National Institutes of Health, Prostate Cancer Foundation, and Scannell Foundation.

C9-methylated quazepam 1 was prepared, and its physicochemical properties were investigated. The atropisomers of 1 were isolated as (a1R, a2S) and (a1S, a2R) isomers. Their absolute configurations were determined based on ECD spectra in comparison with those calculated using the time-dependent density functional theory. Preliminary examination of affinity for the GABAA receptor revealed that the (a1R, a2S) isomer of 1 possessed higher activity than its antipode (a1S, a2R) isomer. The active configuration of C9-methylated quazepam 1 is the same as that of 1,4-benzodiazepin-2-ones.

The Klebsiella oxytoca species complex is part of the human microbiome, especially during infancy and childhood. K. oxytoca species complex strains can produce enterotoxins, namely, tilimycin and tilivalline, while also contributing to colonization resistance (CR). The relationship between these seemingly contradictory roles is not well understood. Here, by coupling ex vivo assays with CRISPR-mutagenesis and various mouse models, we show that K. oxytoca provides CR against Salmonella Typhimurium. In vitro, the antimicrobial activity against various Salmonella strains depended on tilimycin production and was induced by various simple carbohydrates. In vivo, CR against Salmonella depended on toxin production in germ-free mice, while it was largely toxin-independent in mice with residual microbiota. This was linked to the relative levels of toxin-inducing carbohydrates in vivo. Finally, dulcitol utilization was essential for toxin-independent CR in gnotobiotic mice. Together, this demonstrates that nutrient availability is key to both toxin-dependent and substrate-driven competition between K. oxytoca and Salmonella.

BACKGROUND: Among bariatric techniques, sleeve gastrectomy (SG) stands out owing to its efficiency. The role of the stomach as a secretory organ of many substances, such as gastrin, related to insulin secretion is well known. Gastrin induces insulin release in isolated pancreatic islets, limiting somatostatin-14 intraislet release, and has been associated with blood glucose level improvement in diabetic models after SG. SG involves gastric resection along the greater curvature. This study aimed to determine the role of gastrin in glucose metabolism improvement after SG with the aid of the gastrin antagonist netazepide.
METHODS: In 12 sham-operated, 12 SG-operated, and 12 SG-operated/netazepide-treated Wistar rats, we compared medium- and long-term plasma insulin, oral glucose tolerance test (OGTT) results, and plasma gastrin levels. In addition, gastrin expression was assessed in the gastric remnant, and the beta-cell mass was measured.
RESULTS: SG induced a medium-term elevation of the insulin response and plasma gastrin levels without modification of the OGTT results. However, long-term depletion of the insulin response with elevated OGTT areas under the curve and plasma gastrin levels appeared after SG. Netazepide prevented the SG effect on these parameters. Gastrin tissue expression was greater in SG animals than in SG/netazepide-treated or control animals. The beta-cell mass was lower in the SG group than in the control or SG/netazepide group.
CONCLUSIONS: Gastrin plays a central role in glucose improvement after SG. It stimulates a medium-term strong insulin response but also causes long-term beta-cell mass depletion and a loss of insulin response. These effects are prevented by gastrin antagonists such as netazepide.

Hypercholesterolemia is a major risk factor for coronary artery diseases and cardiac ischemic events. Cholesterol per se could also have negative effects on the myocardium, independently from hypercholesterolemia. Previously, we reported that myocardial ischemia-reperfusion induces a deleterious build-up of mitochondrial cholesterol and oxysterols, which is potentiated by hypercholesterolemia and prevented by translocator protein (TSPO) ligands. Here, we studied the mechanism by which sterols accumulate in cardiac mitochondria and promote mitochondrial dysfunction. We performed myocardial ischemia-reperfusion in rats to evaluate mitochondrial function, TSPO, and steroidogenic acute regulatory protein (STAR) levels and the related mitochondrial concentrations of sterols. Rats were treated with the cholesterol synthesis inhibitor pravastatin or the TSPO ligand 4\'-chlorodiazepam. We used Tspo deleted rats, which were phenotypically characterized. Inhibition of cholesterol synthesis reduced mitochondrial sterol accumulation and protected mitochondria during myocardial ischemia-reperfusion. We found that cardiac mitochondrial sterol accumulation is the consequence of enhanced influx of cholesterol and not of the inhibition of its mitochondrial metabolism during ischemia-reperfusion. Mitochondrial cholesterol accumulation at reperfusion was related to an increase in mitochondrial STAR but not to changes in TSPO levels. 4\'-Chlorodiazepam inhibited this mechanism and prevented mitochondrial sterol accumulation and mitochondrial ischemia-reperfusion injury, underlying the close cooperation between STAR and TSPO. Conversely, Tspo deletion, which did not alter cardiac phenotype, abolished the effects of 4\'-chlorodiazepam. This study reveals a novel mitochondrial interaction between TSPO and STAR to promote cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion. This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury.

Mitochondrial dysfunction has been widely implicated in the pathogenesis of Alzheimer\'s disease (AD), with accumulation of damaged and dysfunctional mitochondria occurring early in the disease. Mitophagy, which governs mitochondrial turnover and quality control, is impaired in the AD brain, and strategies aimed at enhancing mitophagy have been identified as promising therapeutic targets. The translocator protein (TSPO) is an outer mitochondrial membrane protein that is upregulated in AD, and ligands targeting TSPO have been shown to exert neuroprotective effects in mouse models of AD. However, whether TSPO ligands modulate mitophagy in AD has not been explored. Here, we provide evidence that the TSPO-specific ligands Ro5-4864 and XBD173 attenuate mitophagy deficits and mitochondrial fragmentation in a cellular model of AD overexpressing the human amyloid precursor protein (APP). Ro5-4864 and XBD173 appear to enhance mitophagy via modulation of the autophagic cargo receptor P62/SQSTM1, in the absence of an effect on PARK2, PINK1, or LC3 level. Taken together, these findings indicate that TSPO ligands may be promising therapeutic agents for ameliorating mitophagy deficits in AD.

Parasitic flatworms cause various clinical and veterinary infections that impart a huge burden worldwide. The most clinically impactful infection is schistosomiasis, a neglected tropical disease caused by parasitic blood flukes. Schistosomiasis is treated with praziquantel (PZQ), an old drug introduced over 40 years ago. New drugs are urgently needed, as while PZQ is broadly effective it suffers from several limitations including poor efficacy against juvenile worms, which may prevent it from being completely curative. An old compound that retains efficacy against juvenile worms is the benzodiazepine meclonazepam (MCLZ). However, host side effects caused by benzodiazepines preclude development of MCLZ as a drug and MCLZ lacks an identified parasite target to catalyze rational drug design for engineering out human host activity. Here, we identify a transient receptor potential ion channel of the melastatin subfamily, named TRPMMCLZ, as a parasite target of MCLZ. MCLZ potently activates Schistosoma mansoni TRPMMCLZ through engagement of a binding pocket within the voltage-sensor-like domain of the ion channel to cause worm paralysis, tissue depolarization, and surface damage. TRPMMCLZ reproduces all known features of MCLZ action on schistosomes, including a lower activity versus Schistosoma japonicum, which is explained by a polymorphism within this voltage-sensor-like domain-binding pocket. TRPMMCLZ is distinct from the TRP channel targeted by PZQ (TRPMPZQ), with both anthelmintic chemotypes targeting unique parasite TRPM paralogs. This advances TRPMMCLZ as a novel druggable target that could circumvent any target-based resistance emerging in response to current mass drug administration campaigns centered on PZQ.

Inhibitor of Apoptosis Proteins (IAPs) are validated targets for cancer therapy, and the deregulation of their activities within the NF-κB pathway correlates with chemoresistance events, even after treatment with IAPs-antagonists in the clinic (Smac-mimetics). The molecule FC2 was identified as a NF-κB pathway modulator in MDA-MB-231 adenocarcinoma cancer cells after virtual screening of the Chembridge library against the Baculoviral IAP Repeat 1 (BIR1) domain of cIAP2 and XIAP. An improved cytotoxic effect is observed when FC2 is combined with Smac-mimetics or with the cytokine Tumor Necrosis Factor (TNF). Here, we propose a library of 22 derivatives of FC2, whose scaffold was rationally modified starting from the position identified as R1. The cytotoxic effect of FC2 derivatives was evaluated in MDA-MB-231 and binding to the cIAP2- and XIAP-BIR1 domains was assessed in fluorescence-based techniques and virtual docking. Among 22 derivatives, 4m and 4p display improved efficacy/potency in MDA-MB-231 cells and low micromolar binding affinity vs the target proteins. Two additional candidates (4b and 4u) display promising cytotoxic effects in combination with TNF, suggesting the connection between this class of molecules and the NF-κB pathway. These results provide the rationale for further FC2 modifications and the design of novel IAP-targeting candidates supporting known therapies.