High grade serous ovarian cancer (HGSOC) is the most lethal gynecological cause of death in women and requires new treatments to help tackle chemoresistance. Rocaglamides, a promising class of anticancer natural products, function as protein translation inhibitors and trigger apoptosis in other types of solid tumors. Didesmethylrocaglamide ((±)-DDR), a derivative of rocaglamide with potent antitumor activity, was synthesized, including three additional rocaglamide derivatives, (±)-DDR01, (±)-DDR03, and (±)-DDR04, to evaluate their cytotoxicity in HGSOC. Using in vitro models, it was determined that (±)-DDR induced cytotoxicity in ovarian cancer cell lines as early as 24 h after application and activated caspase-3, indicating pro-apoptotic activity. In addition, (±)-DDR was cytotoxic in the PE04 and MCF7-ADR (OVCAR8-RES) cell lines that are resistant to cisplatin and paclitaxel, respectively. Evaluation of each enantiomer revealed the minus enantiomer to be ∼18-fold more potent compared to the plus enantiomer in the OVCAR8 cell line. (-)-DDR was further evaluated using an OVCAR8 xenograft model in mice, and a reduction in tumor burden was observed. Its effective cytotoxicity in drug-sensitive and -resistant cell models suggests that (±)-DDR and its corresponding minus enantiomer may have potential as a new therapeutic strategy against HGSOC.

BACKGROUND:Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania and remains a significant global health burden due to limited therapeutic options and emerging drug resistance, necessitating the search for alternative therapeutic agents. PURPOSE:Our earlier studies have shown that Pentalinonsterol (Pen), isolated from Pentalinon andrieuxii plant, showed promising antileishmanial activities. However, the effects of Pen on the transcriptomic profile and nuclear factor erythroid 2-related factor 2 (Nrf-2) related alterations in host cells remain unclear. STUDY DESIGN:The present study examines the impact of Pen on the transcriptomic profiles of human macrophages infected with Leishmania major, with a particular focus on its modulation of Nrf-2 pathway. METHODS:Bulk RNA-seq analysis revealed 1245 differentially expressed genes (DEGs), including 678 upregulated and 567 downregulated genes, using a significance threshold of log2 fold change ≥ 2 and a false discovery rate ≤ 0.05. RESULTS:We found significant modulation of key cytokines, chemokines, and immune effector genes, including downregulation of IL-10, CXCL2, and CXCR3, and upregulation of TXN, GSR, SOD1, and CXCL8 indicating a selectively regulated inflammatory response. Pathway enrichment analysis demonstrated that Pen modified transcriptional profiles related to oxidative stress responses, validated by RT-PCR and confirmed by increased nuclear Nrf-2 translocation through biochemical and immunofluorescence assays. CONCLUSION:These findings highlight Pen's potential as a dual action therapeutic, selectively modulating immune responses in favor of pathogen control while activating Nrf-2 driven antioxidant defenses to counteract oxidative stress. This integrated activity supports Pen's further advancement as a novel host directed therapeutic for leishmaniasis and other Nrf-2 associated inflammatory conditions.

Lenacapavir (LEN) is a new, first-in-class, long acting, HIV-1 capsid (CA)-targeting inhibitor for treating multidrug-resistant HIV-1 infections. LEN exhibits high potency against all major HIV-1 subtypes including variants resistant to current antiretroviral therapies providing a life-saving opportunity for heavily treatment-experienced adults with multidrug-resistant HIV-1. Despite this, LEN has a relatively low barrier to viral resistance. Clinical trials identified resistance-associated mutations near LEN binding site, with the M66I variant exhibiting highest level of resistance (>3200-fold). These findings necessitate continuing efforts to develop next-generation inhibitors against emerging LEN-resistant mutation. We focused on identifying LEN structural functionalities amenable to modifications and to develop LEN analogs with improved antiviral activity against the M66I mutant. Here, we report a new LEN analog, KFA-027, with substantially improved antiviral activity (EC50 ∼ 444 nM, >20-fold) against M66I variant. Overall, these findings suggest a route for developing next-generation LEN analogs against WT and emerging drug-resistant CA mutations.

The tropical plant Aglaia perviridis is known to produce cyclopenta[b]benzofuran and other types of rocaglate derivatives including silvestrol (3) and 5‴-episilvestrol (4) that are of current pharmacological interest. In the present work, further investigation of A. perviridis roots collected in Vietnam has yielded two new rocaglate acetonide derivatives (1 and 2) and a known pentanor-3,4-seco-dammarane triterpenoid (7) found for the first time as a natural product, in addition to four known rocaglates (3-6). The structures of compounds 1 and 2 were confirmed by partial synthesis experiments, and their potential occurrence as extraction artifacts was investigated by targeted selective ion monitoring using UHPLC-MS. All compounds obtained were evaluated against a panel of four human cancer cell lines, in which the six rocaglate derivatives (1-6) tested all showed submicromolar potencies.

Immune surveillance plays a key role in controlling tumor formation and development, and immune cell-based therapies, such as chimeric antigen receptor (CAR)-T cells and CAR-natural killer (NK) cells, have become important for the treatment of cancer. The proton pump (PP), vacuolar H+-ATPase (V-ATPase), acidifies intracellular organelles, pumps protons across the cell plasma membranes, and regulates the activity of various signaling pathways, and thus has been regarded as a potential target for cancer treatment. In addition, V-ATPase plays an important role in cytotoxic T lymphocytes, extracellular vesicle (EV) endocytosis, innate immune responses (IIR), and phagocytosis and hence has the potential to function as a target for the enhancement of immunotherapy. As potent V-ATPase inhibitors, the arylnaphthalene lignans, diphyllin and its derivatives, have exhibited potent antitumor and immunomodulatory activities. The structurally related aryltetralin lignan, podophyllotoxin, has served as a lead compound for both etoposide and teniposide, which have been developed as effective anticancer agents. In the present review, the role of V-ATPase in cancer immunotherapy and the structure-activity relationships (SARs) of diphyllin and its cytotoxic and V-ATPase inhibitory activities and the mechanisms of action are discussed. Also, the promise of diphyllin and its derivatives in the development of new adjuvants for cancer immunotherapies has been proposed.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a five-year survival rate of 12%. Current therapies are often ineffective due to metastasis and an immunosuppressive tumor microenvironment (TME). Activating Transcription Factor 4 (ATF4), a master regulator of cellular stress, is exploited by cancer cells to promote their survival and protect cells from ferroptosis. Prior published work and data reported in our study provide evidence that high ATF4 expression in the PDAC TME correlates with worse overall survival in PDAC patients. Tomatidine, a natural steroidal alkaloid, has been associated with inhibition of ATF4-dependent signaling in multiple disease settings. We hypothesized that tomatidine targets ATF4-dependent signaling leading to ferroptosis mediated cell death in pancreatic cancer. We discovered in vitro tomatidine treatment of pancreatic cancer cells inhibits tumor growth by MTT cell viability assay and reduced the protein expression of p4EBP1/Total 4EBP1 (downstream of ATF4) in a dose dependent manner. In vivo tomatidine treatment (5mg/kg daily intraperitoneally) significantly reduced pancreatic tumor growth. RNA-sequencing of tomatidine treated vs. vehicle treated human and murine pancreatic cancer cells validated targeting of ATF4 related stress regulatory genes. Further, immunofluorescence revealed that ATF4 preferentially localized in the cytoplasm instead of nucleus in presence of tomatidine suggesting tomatidine inhibited nuclear translocation of ATF4, rendering ATF4 ineffective to work as a transcription factor. Further, chromatin immunoprecipitation revealed that tomatidine reduced the transcriptional binding activity of ATF4 with its downstream genes. Tomatidine significantly enhanced gemcitabine chemosensitivity in 3D ECM-hydrogels and in an orthotopic model of pancreatic cancer in vivo. RNA sequencing suggested ferroptosis mediated cell death was a top hit in tomatidine treated pancreatic cancer cells. Therefore, we validated how tomatidine affects the ferroptosis pathway in pancreatic cancer cells by looking at lipid peroxidation via C11 Bodipy staining, immunoblotting for the antioxidant protein Glutathione peroxidase 4 (GPX4) and assessing the changes in the oxygen consumption rate (OCR) using the seahorse assay. Tomatidine treatment was associated with increased lipid peroxidation, decreased GPX4 protein expression, and enhanced mitochondrial biogenesis in PDAC cells. This study highlights a possible novel therapeutic approach utilizing a plant derived metabolite, tomatidine, to target ATF4 activity and cause ferroptosis mediated cancer cell death leading to tumor burden control and chemotherapy sensitization in the PDAC tumor microenvironment. Citation Format: Debasmita Mukherjee, Srija Chakraborty, Lena Bercz, Liliana D’Alesio, Jessica Wedig, Molly A. Torok, Timothy Pfau, Hannah Lathrop, Shrina Jasani, Abigail Guenther, Jake McGue, Daniel Adu-Ampratwum, James R. Fuchs, Timothy L. Frankel, Maciej Pietrzak, Stacey Culp, Anne M. Strohecker, Aleksander Skardal, Thomas A. Mace. Tomatidine inhibits ATF4 activity and induces ferroptosis to limit pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B097.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents that potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into phase 2a clinical trials. Previous cell culture-based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. Although both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect the direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor-mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR-induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared with the WT virus. By rationally modifying PIR, we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.IMPORTANCEAntiretroviral therapies save the lives of millions of people living with HIV (PLWH). However, the evolution of multi-drug-resistant viral phenotypes is a major clinical problem, and there are limited or no treatment options for heavily treatment-experienced PLWH. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a novel class of antiretroviral compounds that work by a unique mechanism of binding to the non-catalytic site on the viral protein and inducing aberrant integrase multimerization. Accordingly, ALLINIs potently inhibit both wild-type HIV-1 and all drug-resistant viral phenotypes that have so far emerged against currently used therapies. Pirmitegravir, a highly potent and safe investigational ALLINI, is currently advancing through clinical trials. Here, we have elucidated the structural and mechanistic bases behind the emergence of HIV-1 integrase mutations in infected cells that confer resistance to pirmitegravir. In turn, our findings allowed us to rationally develop an improved ALLINI with substantially enhanced potency against the pirmitegravir-resistant virus.

Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2-6), carbonate (7-8), sulfonate (9-16), carbamate (17-20), and ether (21-30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1-30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2-8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue (6), which was acylated on the secondary hydroxy at the 11 position.

Inhibition of translation initiation using eIF4A inhibitors like (-)-didesmethylrocaglamide [(-)-DDR] and (-)-rocaglamide [(-)-Roc] is a potential cancer treatment strategy as they simultaneously diminish multiple oncogenic drivers. We showed that human and dog osteosarcoma cells expressed high levels of eIF4A1/2, particularly eIF4A2. Genetic depletion of eIF4A1 and/or 2 slowed osteosarcoma cell growth. To advance preclinical development of eIF4A inhibitors, we demonstrated the importance of (-)-chirality in DDR for growth-inhibitory activity. Bromination of DDR at carbon-5 abolished growth-inhibitory activity, while acetylating DDR at carbon-1 was tolerated. Like DDR and Roc, DDR-acetate increased the γH2A.X levels and induced G2/M arrest and apoptosis. Consistent with translation inhibition, these rocaglates decreased the levels of several mitogenic kinases, the STAT3 transcription factor, and the stress-activated protein kinase p38. However, phosphorylated p38 was greatly enhanced in treated cells, suggesting activation of stress response pathways. RNA sequencing identified RHOB as a top upregulated gene in both DDR- and Roc-treated osteosarcoma cells, but the Rho inhibitor Rhosin did not enhance the growth-inhibitory activity of (-)-DDR or (-)-Roc. Nonetheless, these rocaglates potently suppressed tumor growth in a canine osteosarcoma patient-derived xenograft model. These results suggest that these eIF4A inhibitors can be leveraged to treat both human and dog osteosarcomas.

Covering: 1970 through June of 2023Verticillins are epipolythiodioxopiperazine (ETP) alkaloids, many of which possess potent, nanomolar-level cytotoxicity against a variety of cancer cell lines. Over the last decade, their in vivo activity and mode of action have been explored in detail. Notably, recent studies have indicated that these compounds may be selective inhibitors of histone methyltransferases (HMTases) that alter the epigenome and modify targets that play a crucial role in apoptosis, altering immune cell recognition, and generating reactive oxygen species. Verticillin A (1) was the first of 27 analogues reported from fungal cultures since 1970. Subsequent genome sequencing identified the biosynthetic gene cluster responsible for producing verticillins, allowing a putative pathway to be proposed. Further, molecular sequencing played a pivotal role in clarifying the taxonomic characterization of verticillin-producing fungi, suggesting that most producing strains belong to the genus Clonostachys (i.e., Bionectria), Bionectriaceae. Recent studies have explored the total synthesis of these molecules and the generation of analogues via both semisynthetic and precursor-directed biosynthetic approaches. In addition, nanoparticles have been used to deliver these molecules, which, like many natural products, possess challenging solubility profiles. This review summarizes over 50 years of chemical and biological research on this class of fungal metabolites and offers insights and suggestions on future opportunities to push these compounds into pre-clinical and clinical development.

IntroductionControl of Campylobacter from farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs of Campylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allows Campylobacter to colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO4).MethodsSince mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigate Campylobacter in infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO4 (1 mM) in C. jejuni 81–176, a human isolate which is widely studied.ResultsFurthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO4 for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1–T8) were selected for further studies. These selected eight Tat inhibitors cleared all tested Campylobacter strains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO4in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 μg/mL for 24 h and completely cleared intracellular C. jejuni cells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO4. Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log; p < 0.01) with minimal disruption of microbiota. In silico analyses predicted that T7 has better drug-like properties than T2 inhibitor and might target a key amino acid residue (glutamine 165), which is located in the hydrophobic core of TatC protein.DiscussionThus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to control C. jejuni spread on farms.

Avian pathogenic E. coli (APEC), a causative agent of colibacillosis, is associated with high mortality and morbidity which results in severe economic losses to the poultry industry worldwide. APEC can be transmitted to humans through the consumption of contaminated poultry products. The limited effect of the current vaccines and the advent of drug-resistant strains have necessitated the development of alternative therapies. Previously, we identified 2 small molecules (SMs; [quorum sensing inhibitor; QSI-5] and [growth inhibitor; GI-7]) with high efficacy in vitro and in chickens subcutaneously challenged with APEC O78. Here, we optimized the oral challenge dose of APEC O78 in chickens to mimic the infection in the natural settings, evaluated the efficacy of the GI-7, QSI-5, and combination of GI-7 and QSI-5 (GI7+ QSI-5) in chickens orally infected with APEC, and compared their efficacy to sulfadimethoxine (SDM), an antibiotic currently used to treat APEC. Using the optimized dose of each SM in drinking water, GI-7, QSI-5, GI7+ QSI-5, and SDM were evaluated in chickens challenged with the optimized dose of APEC O78 (1 × 109 CFU/chicken; orally; d 2 of age) and grown on built-up floor litter. Reduction in mortality was 90, 80, 80, and 70% in QSI-5, GI-7+QSI-5, GI-7, and SDM treated groups compared to the positive control (PC), respectively. GI-7, QSI-5, GI-7+QSI-5, and SDM reduced the APEC load in the cecum by 2.2, 2.3, 1.6, and 0.6 logs and in the internal organs by 1.3, 1.2, 1.4, and 0.4 logs compared to PC (P < 0.05), respectively. The cumulative pathological lesions scores were 0.51, 0.24, 0.0, 0.53, and 1.53 in GI-7, QSI-5, GI-7+QSI-5, SDM, and PC groups, respectively. Overall, GI-7 and QSI-5 individually have promising effects as a potential antibiotic-independent approach to control APEC infections in chickens.

Retrosynthesis is a procedure where a target molecule is transformed into potential reactants and thus the synthesis routes can be identified. Recently, computational approaches have been developed to accelerate the design of synthesis routes. In this paper, we develop a generative framework $\mathsf{G^2Retro}$ for one-step retrosynthesis prediction. $\mathsf{G^2Retro}$ imitates the reversed logic of synthetic reactions. It first predicts the reaction centers in the target molecules (products), identifies the synthons needed to assemble the products, and transforms these synthons into reactants. $\mathsf{G^2Retro}$ defines a comprehensive set of reaction center types, and learns from the molecular graphs of the products to predict potential reaction centers. To complete synthons into reactants, $\mathsf{G^2Retro}$ considers all the involved synthon structures and the product structures to identify the optimal completion paths, and accordingly attaches small substructures sequentially to the synthons. Here we show that $\mathsf{G^2Retro}$ is able to better predict the reactants for given products in the benchmark dataset than the state-of-the-art methods.

PDF file - 397K, HAb18G/CD147, pSTAT3 and survivin levels in a panel of human pancreatic cancer cell lines (related to Figure 1, 2A).

PDF file - 147K, Oncomine data analysis of CD147 expression in pancreatic cancer and its co-expression genes (related to the 7th paragraph of Introduction).

Optimization of degrader properties is often a challenge due to their beyond-rule-of-5 nature. Given the paucity of known E3 ligases and the often-limited choice of ligands with varied chemical structures for a given protein target, degrader linkers represent the best position within the chimeric molecules to modify their overall physicochemical properties. In this work, a series of AT7519-based CDK9 degraders was assembled using click chemistry, facilitating the tuning of aqueous solubility and lipophilicity while retaining their linker type and molecular weight. Using chromatographic logD and kinetic solubility experiments, we show that degraders with similar chemical constitution but varied position of the embedded triazole demonstrate different lipophilicity and aqueous solubility properties. Overall, this work highlights the impact of triazole placement on linker composition through application of click chemistry for degrader synthesis and its ability to be used to promote the achievement of favorable physicochemical properties.

PDF file - 61K, The efficiency of HAb18G/CD147 knock-down using pGIPZ and pTRIPZ shRNA vector in human pancreatic cancer cells (related to Figure 1).

PDF file - 793K, Supplementary Figure S1. PSC secrete variable or undetectable levels of PGE2.