Test Compounds Research Articles

Resin acid derivatives: fungicidal properties and prediction of the spectrum of biological activity

Biologically active substances that confer antiviral, anti-tumour and antimicrobial effects, which are found among the components of plant raw materials, as well as the products of their chemical modification, are the subject of considerable research interest. These objects include tricyclic diterpenoids – extractives of wood of abietic and pimaric types (resin acids). The presence of two reaction centres (double bonds and a carboxyl group) in the structure of resin acids opens up a wide range of possibilities for synthesising useful compounds on their basis. One of the most promising areas for the study of resin acids and their derivatives consists assessing their fungicidal properties to inform their introduction into compositions as additives, as well as in the application of protective films to increase the resistance of composite building materials against the aggressive effects of microorganisms. In the present work, the fungicidal activity of the N-phenylimide of maleopimaric acid and its polyfluoroalkyl ethers synthesised by us was evaluated on the cells of the filamentous fungi Aspergillus niger, Alternaria alternata and Penicillium sp. by the rate of colony formation and growth. A comparative analysis of the biological activity of resin acids and their in silico derivatives was performed. According to the AntiBag Pred forecast, the test compounds having the maximum values of the probabilities of the presence and absence of each type of activity are characterised by the manifestation of antibacterial activity in relation to strains of gram-positive bacteria. The results of the predictive model are consistent with previous experimental data. However, AntiFun Pred data related to the calculation of fungicidal activity were not confirmed in vitro.

NRX-101 (D-Cycloserine + Lurasidone) Is Active against Drug-Resistant Urinary Pathogens In Vitro.

D-Cycloserine (DCS) is a broad-spectrum antibiotic that is currently FDA-approved to treat tuberculosis (TB) disease and urinary tract infection (UTI). Despite numerous reports showing good clinical efficacy, DCS fell out of favor as a UTI treatment because of its propensity to cause side effects. NRX-101, a fixed-dose combination of DCS and lurasidone, has been awarded Qualified Infectious Disease Product and Fast Track Designation by the FDA. In this study, we tested NRX-101 against the urinary tract pathogens Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii in cation-adjusted Mueller-Hinton broth (caMHB) and artificial urine media (AUM). Several strains were multidrug resistant. Test compounds were serially diluted in broth/media. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the test compound at which no bacterial growth was observed. DCS exhibited antibacterial efficacy against all strains tested while lurasidone did not appreciably affect the antibacterial action of DCS in vitro. In AUM, the MICs ranged from 128 to 512 mcg/mL for both DCS and NRX-101. In caMHB, MICs ranged from 8 to 1024 mcg/mL for NRX-101 and 32 to 512 mcg/mL for DCS alone. Our data confirm that DCS has antibacterial activity against reference and drug-resistant urinary pathogens. Furthermore, lurasidone does not interfere with DCS's antimicrobial action in vitro. These results support the clinical development of NRX-101 as a treatment for complicated urinary tract infections.

Polymer Beads Increase Field Responses to Host Attractants in the Dengue Vector Aedes aegypti.

This study investigates the efficacy of three different olfactory cues - cyclohexanone, linalool oxide (LO), and 6-methyl-5-heptan-2-one (sulcatone) - in attracting Aedes aegypti, the primary vector of dengue, using BG sentinel traps in a dengue-endemic area (urban Ukunda) in coastal Kenya. Two experiments were conducted. Experiment 1 compared solid formulations of the compounds in polymer beads against liquid formulations with hexane as the solvent. CO2-baited traps served as controls. In Experiment 2, traps were baited with each compound in the polymer beads, commercial BG-Lure, and CO2. Our results indicate that CO2-baited traps recorded the greatest Ae. aegypti captures in both Experiment 1 and 2, whereas trap captures with polymer beads and solvent-based treatments were comparable. In experiment 2, polymer bead-based treatments yielded significantly greater female captures, each recording ~ 2-fold more captures than traps baited with the BG-Lure. There was no significant difference, however, between the treatments. Female Ae. aegypti captured in CO2-baited traps were mainly unfed (91%), with fewer gravid mosquitoes (6.4%) compared to traps with test compounds (range; 12.7-21.1%). Male captures were lower in LO and BG-Lure baited traps compared to other treatments. Gravimetric analysis showed LO had a slower release rate compared to other compounds. The findings suggest that host-associated compounds loaded on polymer beads are more effective in trapping Ae. aegypti than commercial BG-Lure and reveal sex-specific differences in mosquito responses. These results have implications for mosquito surveillance and control programs, highlighting the potential for selective trapping strategies.

Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, aphenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Evaluation of wound healing potential of lawsone from Lawsonia innermis and its organometallic complexes in rats

Aim of the studyTo study the utility of Lawsone along with its zinc and copper complexes for wound healing activity using excision and incision wound models in rats. MethodsThe complex of plant nathoquinone from Lawsonia inermis called Lawsone viz Law and the metals viz. copper and zinc were synthesized. Molecular Docking (with GSK 3β protein) and Molecular Dynamic studies were carried out. The wound healing activity was evaluated using excision and incision wound models in Albino Wistar rats. The six groups comprised disease control, vehicle control, and standard, groups treated with Lawsone (Law) and its Zn Complex (ZnLaw) and copper complex (CuLaw). The progress of excision wound healing was monitored through wound contraction and biochemical and antioxidant parameters along with histopathology of granulation tissue, while for incision wounds, breaking and tensile strength were determined. ResultsThe molecular docking studies revealed that ZnLaw has the highest affinity with GSK 3β protein. The Molecular Dynamic study determined the stability of Law and its metal complexes with GSK 3β protein and was found to be stable in the environment. The test compounds Law, ZnLaw, and CuLaw displayed significant (p<0.05) wound contraction. There was a significant (P< 0.01) decrease in the period of epithelisation as compared to vehicle control. ZnLaw showed maximum reduction in the period of epithelisation (16.03±0.942). There was a significant improvement in the antioxidant parameters from the granulation tissues of wounds treated with Law, ZnLaw, and CuLaw. The metal complexes of Law viz. ZnLaw and CuLaw showed a remarkable increase in hydroxyproline and hexosamine content as compared to the control. Histopathological findings indicated re-epithelisation, neovascularization, and increased collagen deposition as compared to the disease control. The incision model also revealed a significant (P< 0.001) increase in the tensile strength and breaking strength of the healed tissue as compared to the control. ConclusionLaw and its Zinc and copper complexes exhibited good potential in healing of both incision and excision wounds.

Abstract 4495: Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells

Abstract Background: The identification of novel compounds that specifically target cancer cells while sparing normal tissues is crucial. The orphan tyrosine kinase-like receptor 1 (ROR1) is one such target expressed exclusively in various human malignancies, including pancreatic cancer, but not in normal adult tissues. Inhibiting ROR1 signaling shows promise in halting pancreatic cancer cell proliferation and invasion. In this study, lead compounds CSF1R-IN-1 and MCULE-8715589337 were found as potential inhibitors of the ROR1 and were further evaluated for its potential effect on pancreatic cancer cell viability. Method: We curated a compound library of 6 million from PubChem, 2 million lead-like compounds from ZINC15, and 2 million compounds from diverse vendors. Employing Glide’s HTVS followed by XP, we refined the selection based on docking scores lower than the reference molecule, Ponatinib. Compounds were shortlisted by pre-simulation MMGBSA calculations with Schrödinger Prime MM-GBSA module, focusing on compounds with energies below -100 kcal/mol. Then we performed MD simulation for 100ns. Post-simulation MMGBSA on the last 1 ns of simulation assessed change in ligands’ binding energies. The selected leads underwent further validation utilizing two pancreatic cancer cell lines, MiaPaCa-2 and PANC-1. Half-maximal inhibitory concentration (IC50) was assessed using MTS cell viability assay. Result: Comparing pre- and post-simulation MMGBSA energies, the reference compound showed increased, while CSF1R-IN-1 and MCULE-8715589337 exhibited decreased energies. The reference ligand's RMSD showed stability with fluctuations of 3 to 3.5 Å, whereas CSF1R-IN-1 and MCULE-8715589337 had stable and lower RMSD values (approximately 3 Å and 2.4 Å). Test compounds, CSF1R-IN-1 and MCULE-8715589337 showed better molecular interactions with ROR1 compared to reference. The reference compound formed hydrogen bonds with ILE555 and ASP633, along with pi-pi stacking with PHE552. CSF1R-IN-1 forms a hydrogen bond with ILE555 and ASP633, pi-pi stacking with PHE552, and interaction with SER632 through a water bridge. Additionally, compound MCULE-8715589337 forms hydrogen bonds with ASP633, SER632, LYS506, and GLU523. CSF1R-IN-1 notably inhibited proliferation in MiaPaCa-2 cells (IC50: 1.29 μM) while no efficacy in PANC-1 cells (IC50: 78.41 μM). MCULE-8715589337 showed relatively similar IC50 values of 39.06 μM and 37.73 μM in MiaPaCa-2 and PANC-1 cells, respectively. Conclusion: The reduction in MMGBSA energies, low RMSD values, and a higher number of interactions highlight the better characteristics of both compounds compared to the reference. Our findings show CSF1R-IN-1 and MCULE-8715589337 have efficacy in pancreatic cancer cells, but further studies are required to validate and to understand the mechanism of action. Citation Format: Indrakant Kumar Singh, Shradheya RR Gupta, Pooja Mittal, Shivani Soni, Jae Ho Lo, Francesca Battaglin, Yan Yang, Lesly Torres-Gonzalez, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Heinz Josef Lenz. Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4495.

Abstract 3900: A high-throughput platform identifies novel drug combinations towards acute myeloid leukemia therapy

Abstract Molecularly targeted therapies have reshaped oncology in recent decades, though not all patients are eligible for such treatments and resistance mechanisms limit clinical benefit. In response, combination therapies, which can induce potent synthetic lethalities, bypass drug resistance, and expand treatment options, are increasingly being developed. A major hurdle limiting novel combination therapy discovery is the sheer combinatorics associated with broadly testing two or more agents. Thus, there is a practical need for methodologies that prioritize test compounds and technologies for rapid and efficient screening. Towards these ends, we have developed a novel combination drug screening workflow that integrates machine learning-based drug synergy predictions with a high throughput droplet microfluidics-based screening platform. Our physical system, or FlowMatrix, consists of 9,216 nano wells arrayed in a 96 × 96 grid in which cells are incubated and drug compounds delivered via emulsified droplets along both its rows and columns to facilitate combination screening. Following multi-day incubation, live cells are imaged with fluorescence microscopy and drug combination synergies are calculated from cellular viabilities. In our current setup, which includes four dose drug treatments with replicates and controls, we capture complete data (control, single agent, and 4 × 4 drug-drug combination viability measurements) for up to 100 unique drug combinations within the 9,216 dual droplet loaded wells of one FlowMatrix. Our to-date screening efforts have focused on acute myeloid leukemia (AML) specifically, where we have profiled 12,700 AML cell line-drug combinations in our system. This encompasses &amp;gt;3,700 unique drug combinations profiled in 7 distinct AML cell lines with 183 total matrix runs. Among the strongest hits observed in our screens and confirmed with validation studies (~65% confirmation rate) are combinations currently being tested in clinical trials for other cancers and established therapies paired with non-cancer indicated drugs. In particular, we are interrogating combinations involving CDK12 inhibitors, which affect the transcription of DNA repair proteins, splicing, and cancer cell progression, including unexpected partner drugs that potentiate the effects of CDK12 blockade in AML cell lines. We have uncovered additional unexpected synergistic combinations with KIT inhibitors in RUNX1-RUNX1T1 altered AML lines, ATR inhibitors (including an established combination with Gemcitabine), and the BCL-2 inhibitor Venetoclax, including known partnerships with chemotherapies (e.g. Decitabine and Daunorubicin) and other targeted inhibitors. Deeper studies are underway to further validate and characterize these candidates towards expanding treatment options for AML. Ultimately, our platform may radically enhance drug combination screening potential. Citation Format: Anthony R. Soltis, Boryana Zhelyazkova, Pascal Drane, Efstathios Eleftheriadis, Andrew Ventresco, David A. Weitz, Arlinda Lee, Anthony J. Iafrate. A high-throughput platform identifies novel drug combinations towards acute myeloid leukemia therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3900.

Abstract 6778: New microplate platform with cryopreserved 3D tumor spheroids for faster and convenient pre-clinical testing

Abstract Drug discovery and safety testing depend on predictive in vitro models, including multicellular tissues with organotypic functions. While traditional 2D tumor cell cultures have been extensively used in early drug screening due to their user-friendly nature, automation compatibility, and high throughput, they fall short in representing the intricate 3-dimensional structure, physiology, and complex multicellular nature of actual tumors. However, generating such 3D tissues for each test campaign is labor intensive. In addition, not all researchers have the resources to establish these 3D models. To pave the way, we developed a new platform that offers a range of cryopreserved 3D tumor models in micro plate format allowing for high-content screening. It enables users to start testing compounds within a 3-to-5 days simply by thawing an ARCTis™ plate (Always Ready Cryo Tissues) and following clear instructions. Another advantage of these cryopreserved 3D models derives from the fact that they are made in larger batches which means the start point for compound testing is always the same. This improves data quality, maximizes the test window length, and makes the data comparable across work groups. Currently, our growing portfolio consists of 25 tumor cell lines from ATCC representing the most common solid tumors. Their functionality has been determined based on efficacy tests for each cell line with the apoptotic agent Staurosporine. Various cell lines exhibited compact spheroids with a round morphology, less compact aggregates with non-spherical morphology, or loose cell-cell interaction aggregates, which can correlate with their tumor site of origin. Upon treatment with Staurosporine the dose-response profiles indicated growth inhibition, tumor shrinkage, or tumor cell killing effects based on size assessment and cell metabolic analysis (ATP). Moreover, we demonstrated the robust performance of these assays from plates stored for up to 6 months at -80°C.In conclusion, this groundbreaking approach addresses the limitations of traditional 2D cell cultures by providing a high-throughput, in-plate frozen, 3D cell culture platform. By offering a combination of a more physiologically relevant representation of tumors with a robust frozen-storage and time flexible assay platform our method holds great promise for advancing cancer drug discovery by easing the exploration of complex drug testing screens in vitro. Citation Format: Nils Goedecke, Simon Stroebel, Simon Hutter, Tala Issa, Irina Agarkova, Laure-Anne Ligeon, Olivier Frey. New microplate platform with cryopreserved 3D tumor spheroids for faster and convenient pre-clinical testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6778.

Abstract 2920: In vitro assays for investigating the effects of cancer therapeutics on bone

Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2920.

Abstract 90: In vitro functional evaluation of immuno-oncology drug candidates in customized bioassays

Abstract Increased understanding of the complex tumor microenvironment and its interactors has boosted therapeutic interest and highlighted the importance of the development of innovative in vitro bioassays to represent all the players of the cancer immune response and thereby potentially provide pivotal information on the functional dynamics of candidate therapeutics. Regulatory T cells play an important role by downregulating the anti-tumor response. Their regulation mechanisms constitute an important target for new therapeutics. To study these mechanisms in a human model, suppressive Treg bioassays, mimicking the suppressive action of these cells, were developed and optimized. We further highlight the study of neutrophil function in vitro using flow-cytometry or the Incucyte® live imaging system to monitor neutrophil functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), Neutrophil Extracellular Traps (NETs) formation and the release of Reactive Oxygen Species (ROS). Additionally, multiplex cytokine and peroxidase enzyme analysis after activation and stimulation of neutrophils can be evaluated using ELISA or Luminex technology. Moreover, to refine/reduce non-human primate (NHP) usage, we aimed to develop cost-effective NHP in vitro assays with predictive value for in vivo studies. We isolated Cynomolgus Macaque monocytes and successfully differentiated/polarized them into M0- and M2-like macrophages with a cell surface phenotype comparable to that of their human counterparts. We further developed a Cynomolgus Macaque PBMC x human dendritic cell (DC) mixed lymphocyte reaction (MLR) assay for functional evaluation of test compounds, effectively generated Cynomolgus Macaque immature DCs and implemented Cynomolgus Macaque cytokine release assays.Using high quality primary human immune cells from our own biobank, in vitro bioassays have been developed with superior robustness and reproducibility to screen candidate Immuno-Oncology therapeutics. Citation Format: Martijn Vlaming, Emmanuelle Sidot, Jezabel Lefèvre, Ellen Boelen, Sofie Pattyn. In vitro functional evaluation of immuno-oncology drug candidates in customized bioassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 90.

Anticancer Potential and Safety Profile of β-Lapachone In Vitro.

Ipê is a plant of the Bignoniaceae family. Among the compounds extracted from this tree, lapachol is notable because its structural modification allows the production of β-lapachone, which has anticancer properties. The objective of this work was to test this hypothesis at a cellular level in vitro and assess its potential safety for use. The following tests were performed: MTT cell viability assay, apoptotic index determination, comet assay, and micronucleus test. The results showed that β-lapachone had a high cytotoxic capacity for all cell lines tested: ACP02 (gastric adenocarcinoma cells), MCF7 (breast carcinoma cells), HCT116 (colon cancer cells) and HEPG2 (hepatocellular carcinoma cells). Regarding genotoxicity, the exposure of cells to sublethal doses of β-lapachone induced DNA damage (assessed by the comet assay) and nuclear abnormalities, such as nucleoplasmic bridges and nuclear buds (assessed by the micronucleus test). All tested cell lines responded similarly to β-lapachone, except for ACP02 cells, which were relatively resistant to the cytotoxic effects of the compound in the MTT test. Our results collectively indicate that although β-lapachone showed antiproliferative activity against cancer cell lines, it also caused harmful effects in these cells, suggesting that the use of β-lapachone in treating cancer should be carried out with caution.

Protective effect of sucrose esters from cape gooseberry (Physalis peruviana L.) in TNBS-induced colitis.

Phytotherapy is an attractive strategy to treat inflammatory bowel disease (IBD) that could be especially useful in developing countries. We previously demonstrated the intestinal anti-inflammatory effect of the total ethereal extract from the Physalis peruviana (Cape gooseberry) calyces in TNBS-induced colitis. This work investigates the therapeutic potential of Peruviose A and B, two sucrose esters that constitute the major metabolites of its calyces. The effect of the Peruvioses A and B mixture on TNBS-induced colitis was studied after 3 (preventive) and 15-days (therapy set-up) of colitis induction in rats. Colonic inflammation was assessed by measuring macroscopic/histologic damage, MPO activity, and biochemical changes. Additionally, LPS-stimulated RAW 264.7 macrophages were treated with test compounds to determine the effect on cytokine imbalance in these cells. Peruvioses mixture ameliorated TNBS-induced colitis in acute (preventive) or established (therapeutic) settings. Although 3-day treatment with compounds did not produce a potent effect, it was sufficient to significantly reduce the extent/severity of tissue damage and the microscopic disturbances. Beneficial effects in the therapy set-up were substantially higher and involved the inhibition of pro-inflammatory enzymes (iNOS, COX-2), cytokines (TNF-α, IL-1β, and IL-6), as well as epithelial regeneration with restoration of goblet cells numbers and expression of MUC-2 and TFF-3. Consistently, LPS-induced RAW 264.7 cells produced less NO, PGE2, TNF-α, IL-6, and MCP-1. These effects might be related to the inhibition of the NF-κB signaling pathway. Our results suggest that sucrose esters from P. peruviana calyces, non-edible waste from fruit production, might be useful as an alternative IBD treatment.

Design, Synthesis, and Biological Evaluation of Novel Coumarin Analogs Targeted against SARS-CoV-2.

SARS-CoV, an RNA virus, is contagious and displays a remarkable degree of adaptability, resulting in intricate disease presentations marked by frequent genetic mutations that can ultimately give rise to drug resistance. Targeting its viral replication cycle could be a potential therapeutic option to counter its viral growth in the human body leading to the severe infectious stage. The Mpro of SARS-CoV-2 is a promising target for therapeutic development as it is crucial for viral transcription and replication. The derivatives of β-diketone and coumarin have already been reported for their antiviral potential and, thus, are considered as a potential scaffold in the current study for the computational design of potential analogs for targeting the viral replication of SARS-CoV-2. In our study, we used novel diketone-hinged coumarin derivatives against the SARS-CoV-2 MPro to develop a broad-spectrum antiviral agent targeting SARS-CoV-2. Through an analysis of pharmacokinetics and docking studies, we identified a list of the top 10 compounds that demonstrated effectiveness in inhibiting the SARS-CoV-2 MPro virus. On the basis of the pharmacokinetics and docking analyses, the top 5 novel coumarin analogs were synthesized and characterized. The thermodynamic stability of compounds KS82 and KS94 was confirmed by their molecular dynamics, and the stability of the simulated system indicated their inhibitory nature. Molecules KS82 and KS94 were further evaluated for their anti-viral potential using Vero E6 cells followed by RT-PCR assay against SARS-CoV-2. The test compound KS82 was the most active with the potential to inhibit SARS-CoV-2 replication in Vero E6 cells. These data indicate that KS82 prevents the attack of the virus and emerges as the primary candidate with promising antiviral properties.

Antiemetic activity of abietic acid possibly through the 5HT3 and muscarinic receptors interaction pathways

The present study was designed to evaluate the antiemetic activity of abietic acid (AA) using in vivo and in silico studies. To assess the effect, doses of 50 mg/kg b.w. copper sulfate (CuSO4⋅5H2O) were given orally to 2-day-old chicks. The test compound (AA) was given orally at two doses of 20 and 40 mg/kg b.w. On the other hand, aprepitant (16 mg/kg), domperidone (6 mg/kg), diphenhydramine (10 mg/kg), hyoscine (21 mg/kg), and ondansetron (5 mg/kg) were administered orally as positive controls (PCs). The vehicle was used as a control group. Combination therapies with the referral drugs were also given to three separate groups of animals to see the synergistic and antagonizing activity of the test compound. Molecular docking and visualization of ligand-receptor interaction were performed using different computational tools against various emesis-inducing receptors (D2, D3, 5HT3, H1, and M1–M5). Furthermore, the pharmacokinetics and toxicity properties of the selected ligands were predicted by using the SwissADME and Protox-II online servers. Findings indicated that AA dose-dependently enhances the latency of emetic retching and reduces the number of retching compared to the vehicle group. Among the different treatments, animals treated with AA (40 mg/kg) exhibited the highest latency (98 ± 2.44 s) and reduced the number of retching (11.66 ± 2.52 times) compared to the control groups. Additionally, the molecular docking study indicated that AA exhibits the highest binding affinity (− 10.2 kcal/mol) toward the M4 receptors and an elevated binding affinity toward the receptors 5HT3 (− 8.1 kcal/mol), M1 (− 7.7 kcal/mol), M2 (− 8.7 kcal/mol), and H1 (− 8.5 kcal/mol) than the referral ligands. Taken together, our study suggests that AA has potent antiemetic effects by interacting with the 5TH3 and muscarinic receptor interaction pathways. However, additional extensive pre-clinical and clinical studies are required to evaluate the efficacy and toxicity of AA.

Hepatitis A Virus Infection in Cynomolgus Monkeys Confounds the Safety Evaluation of a Drug Candidate.

In a 3-month toxicity study in cynomolgus monkeys at a European contract laboratory, animals were infected with HAV, initially resulting in hepatic injury being incorrectly attributed to the test compound. Elevated serum ALT/AST/GLDH (5- to 10-fold) were noted in individual animals from all groups including controls, with no apparent dose, exposure, or time-related relationship. Liver histopathology revealed minimal to slight inflammatory cell accumulation in periportal zones of most animals, and minimal to slight hepatocyte degeneration/necrosis in 10/42 animals from all groups. As these findings were more pronounced in 6 drug-treated animals, including 2/6 in the low dose group, the draft report concluded: "treatment-related hepatotoxicity at all dose levels precluded determination of a NOAEL." However, the unusual pattern of hepatotoxicity suggested a factor other than drug exposure might have caused the hepatic effects. Therefore, snap-frozen liver samples were tested for hepatitis viruses using a PCR method. Tests for hepatitis B, C, and E virus were negative; however, 20/42 samples were positive for hepatitis A virus (HAV). Infection was strongly associated with increased serum ALT/GLDH, and/or hepatocyte degeneration/necrosis. Re-evaluation of the study in light of these data concluded that the hepatic injury was not drug-related. A subsequent 6-month toxicology study in HAV-vaccinated cynomolgus monkeys confirmed the absence of hepatotoxicity. Identification of HAV infection supported progression of the drug candidate into later clinical trials. Although rarely investigated, subclinical HAV infection has occasionally been reported in laboratory primates, including those used for toxicology studies and it may be more prevalent than the literature indicates.

Relationship between prediction accuracy and uncertainty in compound potency prediction using deep neural networks and control models

The assessment of prediction variance or uncertainty contributes to the evaluation of machine learning models. In molecular machine learning, uncertainty quantification is an evolving area of research where currently no standard approaches or general guidelines are available. We have carried out a detailed analysis of deep neural network variants and simple control models for compound potency prediction to study relationships between prediction accuracy and uncertainty. For comparably accurate predictions obtained with models of different complexity, highly variable prediction uncertainties were detected using different metrics. Furthermore, a strong dependence of prediction characteristics and uncertainties on potency levels of test compounds was observed, often leading to over- or under-confident model decisions with respect to the expected variance of predictions. Moreover, neural network models responded very differently to training set modifications. Taken together, our findings indicate that there is only little, if any correlation between compound potency prediction accuracy and uncertainty, especially for deep neural network models, when predictions are assessed on the basis of currently used metrics for uncertainty quantification.

Soil-specific outcomes in the OECD 216 Nitrogen Transformation Test.

The Organisation for Economic Co-operation and Development (OECD) 216 test guideline investigates the impact of agrochemicals on soil nitrogen transformation. After an evaluation of 465 OECD 216 studies, we describe two distinct yet contrasting outcomes in control nontreated samples that are possible in this testing framework, which we term the "rise" (consistent increases in nitrate concentrations throughout the test period) and "dip" (initial decline in nitrate concentration between Days 0-7, followed by a net-generation of nitrate across Days 7-28) responses. We raise significant concerns that control data from standardized, internationally recognized test guidelines can demonstrate such dissimilar patterns. We propose that, when present, the dip response undermines the intended functioning of the test system and removes the ability to draw appropriate ecotoxicological inferences from the data. In this work, we hypothesize the dip response is a product of conducting the study in low nitrogen content soils. Our results indicate that the dip response can be alleviated by using ammonium sulfate as an immediately available inorganic nitrogen source in place of the guideline-mandated complex, organic lucerne meal, demonstrating the influence of nitrogen availability and accessibility. However, not all low nitrogen soils exhibited the dip response, indicating the involvement of additional unidentified factors. Using our data and real-world regulatory examples, we advocate that datasets displaying the dip response should not be considered valid OECD 216 studies due to the influence of soil properties precluding an assessment of whether any impacts observed are driven solely by the test compound in question or are instead a product of the soil used. We propose methods to account for these soil-specific responses that could be integrated into the conduct and interpretation of OECD 216 studies. Such amendments will improve the reliability and robustness of the study system and enhance confidence in ecotoxicological conclusions derived from OECD 216 datasets. Integr Environ Assess Manag 2024;20:1611-1624. © 2024 SETAC.

Molecular Insights into the Anticancer Mechanism of Glycyrrhetinic Acid in Hepatocellular Carcinoma

The study aimed to predict the binding affinity and interaction patterns between DNMT1, DNMT2, DNMT3A, DNMT3B, TET-1, c-Myc, TET-2, NF-kB and methionine synthase in complex with Glycyrrhetinic acid (GA) using molecular docking simulations. In this study, A crystal structure of proteins (DNA methyltransferase 1, DNA methyltransferase 2, DNA methyltransferase 3A, DNA methyltransferase 3B, NF-kB, TET-1, c-Myc, TET-2 and methionine synthetase) was downloaded from the Protein Data Bank (PDB). the Auto Dock Vina and visualization by Discovery Studio and Chimera program were utilized for molecular docking study. The docking findings are examined to determine the docking pose based on binding affinity, hydrogen bonding, and other beneficial interactions (hydrophobic bond). In addition, it is used to visualise the proteins ligand interactions and analyze the binding pose of GA. Comparing the various binding energies and torsions of the test compound and the control revealed that the test GA had a perfect docking score, and it was predicted to possess comparable anti-tumour and anticancer activity.

Efficient retrosynthetic planning with MCTS exploration enhanced A* search

Retrosynthetic planning, which aims to identify synthetic pathways for target molecules from starting materials, is a fundamental problem in synthetic chemistry. Computer-aided retrosynthesis has made significant progress, in which heuristic search algorithms, including Monte Carlo Tree Search (MCTS) and A* search, have played a crucial role. However, unreliable guiding heuristics often cause search failure due to insufficient exploration. Conversely, excessive exploration also prevents the search from reaching the optimal solution. In this paper, MCTS exploration enhanced A* (MEEA*) search is proposed to incorporate the exploratory behavior of MCTS into A* by providing a look-ahead search. Path consistency is adopted as a regularization to improve the generalization performance of heuristics. Extensive experimental results on 10 molecule datasets demonstrate the effectiveness of MEEA*. Especially, on the widely used United States Patent and Trademark Office (USPTO) benchmark, MEEA* achieves a 100.0% success rate. Moreover, for natural products, MEEA* successfully identifies bio-retrosynthetic pathways for 97.68% test compounds.

MOLECULAR DOCKING OF THE KERUING's (DIPTEROCARPUS) GENUS, SECONDARY METABOLITES OF THE DIPTEROCARPACEAE FAMILY'S AS ANTI-INFLAMMATION AGAINST CYCLOOXYGENASE-2 (COX-2)

Objective: Kalimantan, Indonesia, has a tropical forest abundant in forest products. One of these products is the Dipterocarp tree, which includes the Keruing genus (Dipterocarpus). Dipterocarpus contains secondary metabolites that may be potential sources for new drug compounds. One of these metabolites has the potential to act as an anti-inflammatory agent. Based on pharmacophore modelling and molecular docking, this study used molecular docking to investigate the inhibitory mechanism and affinity of Dipterocarpus secondary metabolites on the 3N8Y inflammatory receptor. Methods: The study involved multiple stages, such as preparing and optimizing the structure of the test compounds, constructing a 3D receptor structure of 3N8Y, validating the methodology, and performing energy docking simulations to analyze the interactions. From the study that has been done, the results for the test compounds were evaluated for their MolDockScore, Pharmacokinetic parameters (ADME), and toxicity. Results: The results revealed that the oligomer resveratrol compound exhibited the lowest MolDockScore value of-104.7400, comparable to natural ligands. In addition to that, this method produces reliable outcomes through pharmacokinetic predictions such as HIA (88.4794%), Caco2 (5.1917 nm/sec), and PPB (100%). Furthermore, the toxicity profile exhibited negative results for mutagenic, non-mutagenic, and carcinogenic tests, including genotoxic and nongenotoxic substances. Conclusion: The oligomeric resveratrol (3',5',4-trihidroksi-trans-stilben) compounds have potential as anti-inflammatory agents by acting on the 3N8Y receptor, which further needs to be tested in vivo.