Enzyme Inhibitors Research Articles

Exploring the potential of semi-synthetic Swertiamarin analogues for GLUT facilitation and insulin secretion in NIT-1 cell lines: a molecular docking and in-vitro study

Synthesis, characterisation, and anti-diabetic potential of swertiamarin analogues against DPP-4 enzymatic inhibition was done prior to this study. However, swertiamarin and its analogues inhibited DPP-4 enzyme significantly. Semisynthetic swertiamarin analogues have been studied for antidiabetic potential and mechanism of action utilising molecular docking and in-vitro techniques. The mechanism of action for swertiamarin analogues was determined by in-silico molecular docking studies using glucose-transporters, GLUT-1 (PDB ID: 4PYP), GLUT-3 (PDB ID: 7SPS), and GLUT-4 (PDB ID: 7WSM) along with in-vitro glucose uptake and glucose-induced insulin secretion assays. These studies found that synthesised swertiamarin analogues SNIPERSV3, SNIPERSV4, and SNIPERSV7 shown better docking score against different GLUTs and better anti-diabetic effects on glucose uptake and insulin secretion in NIT-1 cell line than standard glibenclamide and swertiamarin. Thus, swertiamarin analogues might be studied for diabetes therapy in the future.

Nanoemulsion containing Jatropha gossypiifolia leaf extract reduces dermonecrosis induced by Bothrops erythromelas venom and accelerates wound closure

Ethnopharmacological relevanceThe species Jatropha gossypiifolia, popularly known as “pinhão-roxo”, is distributed throughout Brazil, is commonly employed for topical or oral administration in treating wounds, inflammations, and snake bites. Given the significant impact of snakebites on public health and the limitations of antivenom, coupled with the diverse molecular composition of this plant species, investigating its healing and antidermonecrotic capacities is relevant. Aim of the studyThis study aimed to develop a topical nanoemulsion incorporating the hydroethanolic extract of J. gossypiifolia leaves, to evaluate its therapeutic potential, particularly in terms of its efficacy in wound healing and inhibition of dermonecrosis induced by B. erythromelas venom (BeV). Material and methodsThe extract of J. gossypiifolia (JgE) leaves was obtained by maceration and remaceration. The phytochemical analysis was conducted and J. gossypiifolia nanoemulsion (JgNe) was obtained, characterized and assessed for stability. The cytotoxicity was determined in normal cells (erythrocytes and 3T3) using hemolytic assay and cell viability assay using crystal violet staining. The antioxidant activity was evaluated by the reduction of ABTS and DPPH radicals. The evaluation of wound healing was conducted in vivo following treatment with JgNe, wherein the percentage of wound closure and inflammatory mediators. The skin irritation test was assessed in vivo by applying JgNe directly to the animal's skin. In vitro, the antivenom capacity was evaluated through enzymatic inhibition assays (phospholipase A2 and hyaluronidase) of BeV. Additionally, the in vivo antidermonecrotic activity of JgNe was evaluated by measuring the reduction of the dermonecrotic halo. ResultsThe HPLC-DAD analysis identified flavonoids, specifically vitexin, luteolin derivatives and apigenin derivatives. In addition, 95.08 ± 5.46 mg of gallic acid/g of extract and 137.92 ± 0.99 mg quercetin/g extract, was quantified. JgNe maintained stability over a 4-week period. Moreover, JgE and JgNe demonstrated no cytotoxicity in human erythrocytes and murine fibroblasts at tested concentrations (32.25–250 μg/mL). Additionally, exhibited significant antioxidant activity by reducing ABTS and DPPH radicals. The treatment with JgNe did not induce skin irritation and accelerated wound healing, with significant wound closure observed from 5th day and reduction in nitrite levels, myeloperoxidase activity, and cytokine. Both JgE and JgNe demonstrated in vitro inhibition of the phospholipase and hyaluronidase enzymes of BeV. Moreover, JgNe exhibited antidermonecrotic activity by reducing the dermonecrotic halo caused by BeV after 24 h. ConclusionsJgNe and JgE exhibited no cytotoxicity at the tested concentrations. Additionally, our findings demonstrate that JgNe has the ability to accelerate wound closure and reduce dermonecrosis caused by BeV, indicating to be promising formulation for complementary therapy to antivenom treatment.

Design, Synthesis, Characterization, Enzyme Inhibition, Molecular Docking, and Pharmacological Evaluation of New Chalcone‐Sulfonate Derivatives Bearing Thiophene

AbstractThe novel chalcone‐sulfonate derivatives bearing thiophene motif were synthesized and characterized using 1H NMR, 13C NMR, and HRMS analysis. The evaluation of in vitro and in silico potential pancreatic lipase inhibition activity of the novel chalcone‐sulfonate derivatives bearing thiophene motif was scanned. IC50 values of compounds 5 i (28.76±2.11 μM) and 5 f (30.58±0.45 μM) were determined to be more effective pancreatic lipase inhibitors for in vitro studies. The best potential inhibitor for pancreatic lipase binding affinity was found as compound 5 f (−9.8 kcal mol−1) for in silico studies. Although compounds 5 f and 5 i were identified as the best pancreatic lipase inhibitor candidates in vitro and molecular docking studies, compounds 5 f and 5 i were predicted mutagenic and carcinogenic properties in mice according to ADMET studies. Deeply, compound 5 h was a more effective pancreatic lipase inhibitor according to enzyme inhibition, molecular docking, and ADMET studies. It can be said that compound 5 h may be a more efficient drug candidate than orlistat in the treatment of obesity.

Determination of chemical composition and antioxidant, cytotoxic, antimicrobial, and enzyme inhibition activities of Rumex acetosella L. plant extract

Determination of chemical composition and antioxidant, cytotoxic, antimicrobial, and enzyme inhibition activities of Rumex acetosella L. plant extract

Efficient and knowledge-based hierarchal virtual screening applied to identify potential inhibitors of cholinesterase enzyme

Efficient and knowledge-based hierarchal virtual screening applied to identify potential inhibitors of cholinesterase enzyme

Inhibitory activity of luteolin on α‐glucosidase and pancreatic lipase in vitro and in vivo

SummaryObesity is becoming a top global health problem. Inhibition of digestive enzymes is an effective approach in weight management due to the reduction of energy absorption. In this study, the inhibitory effects of luteolin on α‐glucosidase and pancreatic lipase were investigated in vitro and in vivo via enzyme kinetics, fluorescence quenching, molecular docking and rat experiments. The results showed that luteolin inhibited α‐glucosidase and pancreatic lipase in a mixed‐type and uncompetitive manner with IC50 values of 29.31 and 42.74 μg mL−1 respectively. The binding of luteolin with the two enzymes caused the quenching of intrinsic fluorescence of protein. Molecular docking simulation revealed that luteolin could be located in the active pocket of α‐glucosidase and interact with the key amino acid residues. Moreover, a hydrophobic interaction and a van der Waals force were formed between luteolin and the key amino acid residues of pancreatic lipase (His264 and Ser153). In vivo studies further confirmed that luteolin could significantly decrease the absorption of glucose and triacylglyceride in rats by inhibiting α‐glucosidase and pancreatic lipase.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Exploring the multifaceted bioactivities of Lavandula pinnata L. essential oil: promising pharmacological activities.

Introduction: This study investigates the biological activities of Lavandula pinnata essential oil (LPEO), an endemic lavender species from the Canary Islands, traditionally used in treating various ailments. Methods: LPEO was extracted by hydrodistillation and analyzed using GC-MS. Antioxidant activity was assessed by DPPH radical scavenging and total antioxidant capacity assays. Antimicrobial activity was evaluated by disc diffusion, MIC, MBC, and MFC determination against bacterial (Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa) and fungal (Candida glabrata, Rhodotorula glutinis, Aspergillus niger, Penicillium digitatum) strains. Antidiabetic and anti-gout potential were investigated through α-amylase, α-glucosidase, and xanthine oxidase inhibition assays. Antityrosinase activity was determined using a modified dopachrome method. Cytotoxicity was assessed by MTT assay against breast (MCF-7, MDA-MB-468), liver (HepG2), colon (HCT-15) cancer cells, and normal cells (PBMCs). Results and discussion: LPEO exhibits potent antiradical activity (IC50 = 148.33 ± 2.48μg/mL) and significant antioxidant capacity (TAC = 171.56 ± 2.34μg AA/mg of EO). It demonstrates notable antibacterial activity against four strains (Staphylococcus aureus, Micrococcus luteus, Escherichia coli, and Pseudomonas aeruginosa) with inhibition zones ranging from 18.70 ± 0.30mm to 29.20 ± 0.30mm, along with relatively low MIC and MBC values. LPEO displays significant antifungal activity against four strains (Candida glabrata, Rhodotorula glutinis, Aspergillus niger, and Penicillium digitatum) with a fungicidal effect at 1mg/mL, surpassing the positive control (cycloheximide), and MIC and MFC values indicating a fungicidal effect. It exhibits substantial inhibition of xanthine oxidase enzyme (IC50 = 26.48 ± 0.90μg/mL), comparable to allopurinol, and marked inhibitory effects on α-amylase (IC50 = 31.56 ± 0.46μg/mL) and α-glucosidase (IC50 = 58.47 ± 2.35μg/mL) enzymes.The enzyme tyrosinase is inhibited by LPEO (IC50 = 29.11 ± 0.08mg/mL). LPEO displays moderate cytotoxic activity against breast, liver, and colon cancer cells, with low toxicity towards normal cells (PBMC). LPEO exhibits greater selectivity than cisplatin for breast (MCF-7) and colon (HCT-15) cancer cells but lower selectivity for liver (HepG2) and metastatic breast (MDA-MB-468) cancer cells. These findings suggest the potential of LPEO as an antioxidant, antimicrobial, anti-gout, antidiabetic, and anticancer agent.

Approaches in Managing Resistant Hypertension: A Review.

In India, around 234 million adults (one in three) suffer from hypertension (HTN). An average of 10% of these cases are likely to be resistant hypertension (RH). This load of 23 million patients is expected to expand further with revisions in diagnostic criteria. The treatment and control rates of hypertension in India average around 30% and 15%, respectively. Pharmacological management involves a stepwise approach starting with optimizing the A-C-D (angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs), and thiazide-like diuretics) triple-drug combination, followed by substitution with a thiazide-like diuretic and use of spironolactone as a next step (fourthdrug). The subsequent steps are suggestions based on expert input and must be individualized. These include using a β-blocker as the fifth drug and an α1-blocker or a peripheral vasodilator as a final option when target blood pressure (BP) values are not achieved. Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are likely to be helpful in managing RH due to their renal and cardiovascular protection as well as mortality benefits. SGLT2i lowers BP independent of the dosage and concomitant anti-hypertensive medications. Patient education and tools to monitor BP and treatment compliance will improve outcomes with these medications. In addition to therapeutic intervention, a preventive approach for RH mandates a need to identify patients at risk and use appropriate preventive and optimal therapy to prevent uncontrolled hypertension in patients with cardiovascular disorders.

The Effect of Essential Oils from Asteraceae Plants on Behavior and Selected Physiological Parameters of the Bird Cherry-Oat Aphid.

Essential oils (EOs), including those from the Asteraceae plants, have been shown to have promising insecticidal activity against a wide range of insect pests. Understanding the mechanism of action of EOs is one of the studied aspects. The present study aimed to evaluate the effect of essential oils from Achillea millefolium, Santolina chamaecyparissus, Tagetes patula and Tanacetum vulgare on the settling and probing behavior of the bird cherry-oat aphid (Rhopalosiphum padi L.). In addition, the effect of the oils on the activity of such enzymes as trypsin, pepsin and α- and β-glucosidase involved in the metabolism of proteins and sugars of the insects was examined. The leaf-choice bioassays demonstrated that the studied EOs limited aphid settling for at least 24 h after the treatment. The application of EOs also inferred with aphid probing behavior by reducing the total probing time and total duration of phloem sap ingestion. Aphids spent more time in the search phase due to an increase in the number and total duration of pathway phases. Moreover, the activity of the studied proteases and glucosidases significantly decreased in R. padi females exposed to the EOs. The enzyme inhibition varied depending on the applied oil and exposure time. Generally, the EOs with stronger deterrent activity also showed higher inhibitory effects. The results suggest that the tested EOs disrupt key digestive processes in R. padi which may be an important factor determining their aphicidal activity.

Phytochemical Profile, Antioxidant, Enzyme Inhibition, Acute Toxicity, In Silico Molecular Docking and Dynamic Analysis of Apis Mellifera Propolis as Antidiabetic Supplement.

This study aims to identify the phytochemical profile of Apis mellifera propolis and explore the potential of its anti-diabetic activity through inhibition of α-amylase (α-AE), α-glucosidase(α-GE), as well as novel antidiabetic compounds of propolis. Apis mellifera propolis extract (AMPE) exhibited elevated polyphenol 33.26±0.17 (mg GAE/g) and flavonoid (15.45±0.13 mg RE/g). It also indicated moderate strong antioxidant activity (IC50 793.09±1.94 μg/ml). This study found that AMPE displayed promising α-AE and α-GE inhibition through in vitro study. Based on LC-MS/MS screening, 18 unique AMPE compounds were identified, with majorly belonging to anthraquinone and flavonoid compounds. Furthermore, in silico study determined that 8 compounds of AMPE exhibited strong binding to α-AE that specifically interacted with its catalytic residue of ASP197. Moreover, 2 compounds exhibit potential inhibition of α-GE, by interacting with crucial amino acids of ARG315, ASP352, and ASP69. Finally, we suggested that 2,7-Dihydroxy-1-(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene and 3(3-(3,4-Dihydroxybenzyl)-7-hydroxychroman-4-one as novel inhibitors of α-AE and α-GE. Notably, these compounds were initially discovered from Apis mellifera propolis in this study. The molecular dynamic analysis confirmed their stable binding with both enzymes over 100 ns simulations. The in vivo acute toxicity assay reveals AMPE as a practically non-toxic product with an LD50 value of 16,050 mg/kg. Therefore, this propolis may serve as a promising natural product for diabetes mellitus treatment.

Comprehensive Assessment of Cardiac Function in Breast Cancer Patients: Integrating Multiparametric Speckle Tracking Imaging and Conventional Echocardiography Parameters.

Purpose The purpose of this study is to comprehensively evaluate the role of different echocardiography parameters in breast cancer patients undergoing chemotherapy. While echocardiography examination with calculation of ejection fraction (EF), is pivotal for patient monitoring, its operator dependence and insensitivity to subtle changes in left ventricular (LV) contractility present challenges. Global longitudinal strain (GLS), derived from speckle tracking, is more sensitive and stable than EF. Our research aimed to delineate supplementary echocardiography measurements beneficial for the cardiological monitoring of breast cancer patients. Methods Patients were followed up with echocardiography at baseline, during, and after the chemotherapy. Conventional echocardiography and multiple speckle tracking imaging parameters including myocardial work index, atrial strain, twist, and automatic EF were investigated. Results A total of 25 patients were recruited. A subset (15/25) exhibited pronounced GLS reduction, associated with decreased EF and altered cardiac mechanics. Patients with unchanged GLS were often hypertensive and on specific medications, in particular angiotensin-converting enzyme inhibitors (ACE inhibitors)/angiotensin II receptor blockers (ARBs), potentially indicating protective effects. Despite stability in other parameters, GLS and EF sensitivity highlight their importance. A strong correlation between manual and automated EF measurement methods was also observed. Conclusion Despite the small sample size, across diverse echocardiography parameters, GLS and EF are primarily affected by chemotherapy. Hypertensive individuals exhibited lower susceptibility to chemotherapy-induced damage, likely attributed to the cardioprotective properties of ACE inhibitors and angiotensin II receptor blockers. A strong correlation between automatic and Simpson-based EF was found.

The influence of obesity on the parameters of the kallikrein-kinin system and proteolysis in chronic biliary pancreatitis

Background. Clinical practice and science have accumulated data that obese patients suffer from severe forms of acute and chronic pancreatitis, which is explained by the accumulation of fat around the pancreas, a decreased activity of pancreatic enzymes. The purpose of the study is to describe the features of the kallikrein-kinin system and proteolysis in chronic biliary pancreatitis (CBP), depending on the presence of comorbid obesity. Materials and methods. One hundred and thirty-seven patients with chronic biliary pancreatitis were examined and divided into two groups depending on the presence of comorbid obesity: group I consisted of 22 patients with CBP and group II — of 115 patients with CBP and obesity. Results. The obtained results proved that an increase in body weight of patients with CBP lead to a more severe kallikrein-kinin system imbalance, with enhanced activation of inflammation and a decrease in the protective effect of the proteolysis. An increase in the degree of obesity in patients with CBP was accompanied by a more severe kallikrein-kinin system and proteolytic imbalance with an increase in the proteolytic enzymes level that have a damaging effect on the hepatic and pancreatic tissues and have a pro-inflammatory activity, as well as by a decrease in the content of the proteolytic enzyme inhibitors, which reliably weakened the protective effect of kallikrein-kinin system and proteolysis. Conclusions. 1) It was proved that there is a higher activity of the kallikrein-kinin system (accor­ding to proteolytic activation and kallikrein levels) and a decrease in the activity of proteolytic enzyme inhibitors (α2-macroglobulin and kininase II) in patients with chronic biliary pancreatitis and comorbid obesity compared to those without obesity (p < 0.05). 2) An increase in the degree of obesity lead to an increase of proteolytic activity and a decrease in the content of the proteolytic enzyme inhibitors in patients with chronic biliary pancreatitis. It was proved the aggravating effect of obesity on the kallikrein-kinin system and proteolytic imbalance, which must be taken into account while forming a comprehensive treatment of such patients.

A new nomogram to predict in-hospital mortality in patients with acute decompensated chronic heart failure and diabetes after 48 Hours of Intensive Care Unit

BackgroundThe study set out to develop an accurate and clinically valuable prognostic nomogram to assess the risk of in-hospital death in patients with acute decompensated chronic heart failure (ADCHF) and diabetes.MethodsWe extracted clinical data of patients diagnosed with ADCHF and diabetes from the Medical Information Mart for Intensive Care III database. Risk variables were selected utilizing least absolute shrinkage and selection operator regression analysis, and were included in multivariate logistic regression and presented in nomogram. bootstrap was used for internal validation. The discriminative power and predictive accuracy of the nomogram were estimated using the area under the receiver operating characteristic curve (AUC), calibration curve and decision curve analysis (DCA).ResultsAmong 867 patients with ADCHF and diabetes, In-hospital death occurred in 81 (9.3%) patients. Age, heart rate, systolic blood pressure, red blood cell distribution width, shock, β-blockers, angiotensin converting enzyme inhibitors or angiotensin receptor blockers, assisted ventilation, and blood urea nitrogen were brought into the nomogram model. The calibration curves suggested that the nomogram was well calibrated. The AUC of the nomogram was 0.873 (95% CI: 0.834–0.911), which was higher that of the Simplified Acute Physiology Score II [0.761 (95% CI: 0.711–0.810)] and sequential organ failure assessment score [0.699 (95% CI: 0.642–0.756)], and Guidelines-Heart Failure score [0.782 (95% CI: 0.731–0.835)], indicating that the nomogram had better ability to predict in-hospital mortality. In addition, the internally validated C-index was 0.857 (95% CI: 0.825–0.891), which again verified the validity of this model.ConclusionsThis study constructed a simple and accurate nomogram for predicting in-hospital mortality in patients with ADCHF and diabetes, especially in those who admitted to the intensive care unit for more than 48 hours, which contributed clinicians to assess the risk and individualize the treatment of patients, thereby reducing in-hospital mortality.

Angiotensin-Converting Enzyme Inhibitors or Angiotensin Receptor Blockers After Transcatheter Aortic Valve Replacement: A Meta-Analysis

BackgroundPersistent left ventricular hypertrophy after transcatheter aortic valve replacement (TAVR) has been associated with poor outcomes. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs), due to their favorable effects on ventricular remodeling, have been hypothesized to improve outcomes post-TAVR, yet there are no recommendations regarding their use. ObjectivesThis study aimed to compare the outcomes of patients receiving ACEIs/ARBs with those not receiving ACEIs/ARBs after TAVR. MethodsWe performed a literature search on PubMed and Cochrane Library until June 14, 2023, and included all studies comparing clinical outcomes between patients given ACEIs/ARBs and those not given ACEIs/ARBs after TAVR. All-cause mortality was the primary outcome. We used a random effects model with appropriate corrections to calculate relative risk (RR) and CIs, with all analyses carried out using R v4.0.3. ResultsWe included ten studies on the use of ACEIs/ARBs post-TAVR. Patients on ACEIs/ARBs had lower risk of all-cause mortality (RR: 0.74, 95% CI: 0.65-0.86, I2 = 62%, chi-square P < 0.01), cardiovascular mortality (RR: 0.70, 95% CI: 0.56-0.88, I2 = 0%, chi-square P = 0.54), and new-onset atrial fibrillation (RR: 0.71, 95% CI: 0.52-0.96, I2 = 0%, chi-square P = 0.59). Patients on ACEIs/ARBs had a similar risk of myocardial infarction, heart failure, stroke, new permanent pacemaker implantation, acute kidney injury, major bleeding, vascular complications, aortic regurgitation, and mitral regurgitation. ConclusionsWe found that patients receiving ACEIs/ARBs had a lower risk of all-cause mortality, cardiovascular mortality, and new-onset atrial fibrillation. Risk of other outcomes was similar to patients not receiving ACEIs/ARBs. Randomized clinical trials are needed to explore the benefits of ACEIs/ARBs post-TAVR, so that definitive guidelines can be developed.

Angiotensin II Is Involved in MLKL Activation During the Development of Heart Failure Following Myocardial Infarction in Rats.

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT1 receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds.

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

Antifungal plant flavonoids identified in silico with potential to control rice blast disease caused by Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, poses a severe threat to rice production, particularly in Asia where rice is a staple food. Concerns over fungicide resistance and environmental impact have sparked interest in exploring natural fungicides as potential alternatives. This study aimed to identify highly potent natural fungicides against M. oryzae to combat rice blast disease, using advanced molecular dynamics techniques. Four key proteins (CATALASE PEROXIDASES 2, HYBRID PKS-NRPS SYNTHETASE TAS1, MANGANESE LIPOXYGENASE, and PRE-MRNA-SPLICING FACTOR CEF1) involved in M. oryzae's infection process were identified. A list of 30 plant metabolites with documented antifungal properties was compiled for evaluation as potential fungicides. Molecular docking studies revealed that 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin exhibited superior binding affinities compared to reference fungicides (Azoxystrobin and Tricyclazole). High throughput molecular dynamics simulations were performed, analyzing parameters like RMSD, RMSF, Rg, SASA, hydrogen bonds, contact analysis, Gibbs free energy, and cluster analysis. The results revealed stable interactions between the selected metabolites and the target proteins, involving important hydrogen bonds and contacts. The SwissADME server analysis indicated that the metabolites possess fungicide properties, making them effective and safe fungicides with low toxicity to the environment and living beings. Additionally, bioactivity assays confirmed their biological activity as nuclear receptor ligands and enzyme inhibitors. Overall, this study offers valuable insights into potential natural fungicides for combating rice blast disease, with 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin standing out as promising and environmentally friendly alternatives to conventional fungicides. These findings have significant implications for developing crop protection strategies and enhancing global food security, particularly in rice-dependent regions.

Abstract LB131: CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization

Abstract Background: CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Immunosupressive adenosine contributes to immune evasion in solid tumors by accumulating within the tumor microenvironment and thereby inhibiting immune effector cells (e.g., CD8+ T cells). CD73 expression levels correlate with a worse prognosis in specific cancer types, including breast cancer. Adenosine production via CD73 can be therapeutically inhibited by two independent mechanisms: (1) enzyme inhibition, and (2) receptor internalization and subsequent degradation. CBO421, a drug Fc-conjugate (DFC), comprised of a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, achieves both mechanisms, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) with potential best-in-class efficacy. Methods: The activities of CBO421, a commercially sourced CD73 small molecule inhibitor, quemliclustat (AB680), and biosimilar anti-CD73 mAb, oleclumab (MEDI9447), were investigated. Enzymatic inhibition of soluble CD73 and membrane-bound CD73 on MDA-MB-231 cells was determined. Receptor-mediated internalization of CD73 was assessed using the human triple-negative breast cancer cell line (TNBC), MDA-MB-231, by flow cytometry. Cells were treated with CBO421 or oleclumab. CD73 internalization was measured as the decrease in CD73 surface receptor signal over a 6 h time-course using an anti-human CD73 detection antibody with non-overlapping epitopes to CBO421 or oleclumab. Results: CD73 inhibition by CBO421 of soluble CD73 or CD73 expressed on the surface of the MDA-MB-231 cell line was comparable or superior to AB680 or oleclumab. Receptor internalization activities of CBO421 and oleclumab were compared. CBO421 and oleclumab internalization percentages were determined at 1 h (52.3% vs 26.7%), 4 h (80.8% vs 38.0%), and 6 h (84.8% vs 42.9%) respectively, with CBO421 demonstrating superior maximal receptor internalization. The unconjugated hIgG1 Fc negative control did not exhibit any receptor-mediated internalization of CD73, as expected. IC50 values were also calculated for CBO421 and oleclumab at 1 h (0.20 nM vs 0.88 nM), 4 h (0.07 nM vs 0.27 nM), and 6 h (0.08 nM vs 0.14 nM), respectively. Conclusions: CBO421 demonstrated potent and complete CD73 enzyme inhibition and robust CD73 receptor internalization in CD73+ cancer cells that was superior to oleclumab, thereby further differentiating CBO421 from existing CD73-targeting therapeutics currently in clinical development. Based on these compelling results and additional emerging data, CBO421 is progressing as a potential best-in-class clinical development candidate for the treatment of solid cancers. Citation Format: Elizabeth Abelovski, Nicholas Dedeic, Simon Döhrmann, James Levin, Amanda Almaguer, Doug Zuill, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421, a novel drug Fc-conjugate, inhibits the enzymatic activity of CD73 and triggers CD73 internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB131.

Abstract CT103: Next generation capecitabine (NGC-Cap) in phase 1b trial significantly increases 5-FU exposure while improving safety profile compared to capecitabine

Abstract Background: Capecitabine (Cape) at the recommended dose of 1,000 - 1,250 mg/m2 bid has been shown to frequently cause clinically meaningful side effects such as myelosuppression and hand-foot syndrome (HFS), both of which may require dose modification, interruption, or discontinuation. HFS is caused by 5-FU catabolites while myelosuppression is caused by 5-FU anabolites. NGC-Cap combines ethynyl-uracil (PCS6422), an irreversible inhibitor of the DPD catabolism enzyme, and Cape. Methods: The Phase 1b trial is a 3+3 design with up to 5 ascending Cape doses from 75 mg qd to 300 mg bid. Cape is given 7 days on/7 days off every 14 days with a single dose of PCS6422 given 16-24 hours before the start of every cycle. The 5-FU AUC(0-9 hrs), Cmax, and T1/2 were calculated on Day 1 when DPD inhibition is at its maximum. New cohorts are opened following a review of the safety data by a cohort review committee after the second cycle. Blood samples are obtained for PK analysis of PCS6422, Cape, and Cape metabolites (e.g., 5-FU, FBAL). All patients have cancer refractory or intolerant to existing available therapies. Radiological tumor response evaluation (RECIST 1.1) is performed every 8 weeks. Results: 18 patients were enrolled in the first 4 dose levels of Cape in NGC-Cap. The 5-FU AUC (geometric mean, CV%) for the 150 and 225 mg bid NGC-Cap cohorts were 3,802 (23%) and 6,311 (37%) ng-hr/ml, respectively. These AUCs were approximately 5-10 times the AUC(0-inf) of 698 (33%) previously reported for a larger dose of approximately 2,250 mg of monotherapy Cape (Mono-Cape) (Reigner 1998). Similarly, the 5-FU Cmaxs (geometric mean, CV%) for these 2 cohorts were greater at 694 (22%) and 1,056 (28%) ng/ml than the Cmax of Mono-Cape at 310 (50%). The 5-FU T1/2 (arithmetic mean, CV%) of 3.54 (18%) and 5.72 (51%) hrs for these two NGC-Cap cohorts were also much longer than the 0.84 (25%) hrs for Mono-Cape. Although 150 and 225 bid NGC-Cap cohorts produced greater Cmax and AUC levels than Mono-Cape, the side effect profile from anabolites for the 150 cohort was better than Mono-Cape while the profile for the 225 cohort was similar to Mono-Cape. The extremely low FBAL catabolite formation and exposure (AUC of &amp;lt; 250 vs 31,400 for Mono-Cape) across all NGC-Cap doses also resulted in only 1 patient having Grade 1 HFS. Conclusion: The Phase 1b trial has already revealed some of the potential benefits of NGC-Cap over Mono-Cap. 1.NGC-Cap can provide a greater 5-FU exposure based on AUC and Cmax with a better or similar side effect profile.2.Side effects from the 5-FU catabolites are minimal and less severe for NGC-Cap.3.Side effects from 5-FU anabolites are dependent on 5-FU exposure with less exposure leading to fewer side effects that may also be less severe.4.NGC-Cap is to be further evaluated in a Phase 2 trial with the expectation that NGC-Cap will provide a better efficacy and safety profile than Cape. Citation Format: David Young, Sian Bigora, Mary Nyberg, Kayla Parks, Amit Mahipar, Patrick Boland, Eric Feldman, Howard Hochster. Next generation capecitabine (NGC-Cap) in phase 1b trial significantly increases 5-FU exposure while improving safety profile compared to capecitabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT103.