Weak Inhibitor Research Articles

First report of piperidone extracts as tyrosinase and α-glucosidase inhibitors from fruits of Piper retrofractum

Importance of the work: Piper retrofractum is used as a spice. This report is the first to identify tyrosinase and α-glucosidase inhibitors extracted from this plant. Objectives: To separate and identify the bioactive compounds from the fruits of P. retrofractum and to evaluate their tyrosinase and α-glucosidase inhibition activities. Materials & Methods: The EtOAc extract was isolated and purified using various chromatographic techniques. The structures of isolated compounds were confirmed based on their spectroscopic data. The tyrosinase and α–glucosidase inhibition activities of the isolated compounds were assessed based on a colorimetric method, using kojic acid and acarbose, respectively, as positive controls. Results: Seven known compounds were identified, named methyl piperate (1), guineensine (2), piperine (3), N-feruloyltyramine (4), (3S, 4S)-4-chloro-3-hydroxy-2-piperidin-2-one (5), 3-chloro-4-hydroxy-2-piperidone (6) and 3α,4α-epoxy-2-piperidone (7). Piperidones 5 and 6 showed tyrosinase inhibition with concentration at 50% inhibition (IC50) values of 4.91 mM and 11.0 mM, respectively, whereas the alkylamides 1–3 and phenylpropanamide 4 were not active. On the other hand, only phenylpropanamide 4 was active against maltase and sucrase with IC50 values of 0.28 mM and 0.5 mM, respectively. Piperidone 5 showed weak inhibition against both maltase and sucrase α-glucosidases, while the piperidones 6 and 7 exhibited weak inhibition against sucrase α-glucosidases. Main finding: The piperidones were more active against tyrosinase than piperine, which is representative metabolite from this plant. Further study on the inhibitory mechanism may lead to suitable applications of the extract in cosmetics.

Discovery of a nasal spray steroid, tixocortol, as an inhibitor of SARS-CoV-2 main protease and viral replication.

Coronaviruses rely on the viral-encoded chymotrypsin-like main protease (Mpro or 3CLpro) for replication and assembly. Our previous research on Mpro of SARS-CoV-2 identified cysteine 300 (Cys300) as a potential allosteric site of Mpro inhibition. Here, we identified tixocortol (TX) as a covalent modifier of Cys300 which inhibits Mpro activity in vitro as well as in a cell-based Mpro expression assay. Most importantly TX inhibited SARS-CoV-2 replication in ACE2 expressing HeLa cells. Biochemical analysis and kinetic assays were consistent with TX acting as a non-competitive inhibitor. By contrast, TX was a weaker inhibitor and modifier of C300S Mpro, confirming a role for Cys300 in inhibition of WT Mpro but also providing evidence for an additional Cys target. TX pivalate (TP), a prodrug for TX that was previously marketed as a nasal spray, also inhibited SARS-CoV-2 replication in HeLa-ACE2 cells at low micromolar IC50s. These studies suggest that TX and/or TP could possibly be repurposed for the prevention and/or treatment of SARS-CoV-2 infection.

Anticholinesterase and Antityrosinase Secondary Metabolites from the Fungus Xylobolus subpileatus.

Xylobolus subpileatus is a widely distributed crust fungus reported from all continents except Antarctica, although considered a rare species in several European countries. Profound mycochemical analysis of the methanol extract of X. subpileatus resulted in the isolation of seven compounds (1-7). Among them, (3β,22E)-3-methoxy-ergosta-4,6,814,22-tetraene (1) is a new natural product, while the NMR assignment of its already known epimer (2) has been revised. In addition to a benzohydrofuran derivative fomannoxin (3), four ergostane-type triterpenes 4-7 were identified. The structure elucidation of the isolated metabolites was performed by one- and two-dimensional NMR and MS analysis. Compounds 2-7 as well as the chloroform, n-hexane, and methanol extracts of X. subpileatus were evaluated for their tyrosinase, acetylcholinesterase, and butyrylcholinesterase inhibitory properties. Among the examined compounds, only fomannoxin (3) displayed the antityrosinase property with 51% of inhibition, and the fungal steroids proved to be inactive. Regarding the potential acetylcholinesterase (AChE) inhibitory activity of the fungal extracts and metabolites, it was demonstrated that the chloroform extract and compounds 3-4 exerted noteworthy inhibitory activity, with 83.86 and 32.99%, respectively. The butyrylcholinesterase (BChE) inhibitory assay revealed that methanol and chloroform extracts, as well as compounds 3 and 4, exerted notable activity, while the rest of the compounds proved to be only weak enzyme inhibitors. Our study represents the first report on the chemical profile of basidiome of the wild-growing X. subpileatus, offering a thorough study on the isolation and structure determination of the most characteristic biologically active constituents of this species.

Structural Studies of Klebsiella pneumoniae Fosfomycin-Resistance Protein and Its Application for the Development of an Optical Biosensor for Fosfomycin Determination.

Fosfomycin-resistance proteins (FosAs) are dimeric metal-dependent glutathione transferases that conjugate the antibiotic fosfomycin (Fos) to the tripeptide glutathione (γ-Glu-Cys-Gly, GSH), rendering it inactive. In the present study, we reported a comparative analysis of the functional features of two FosAs from Pseudomonas aeruginosa (FosAPA) and Klebsiella pneumoniae (FosAKP). The coding sequences of the enzymes were cloned into a T7 expression vector, and soluble active enzymes were expressed in E. coli. FosAKP displayed higher activity and was selected for further studies. The crystal structure of the dimeric FosAKP was determined via X-ray crystallography at 1.48 Å resolution. Fos and tartrate (Tar) were found bound in the active site of the first and second molecules of the dimer, respectively. The binding of Tar to the active site caused slight rearrangements in the structure and dynamics of the enzyme, acting as a weak inhibitor of Fos binding. Differential scanning fluorimetry (DSF) was used to measure the thermal stability of FosAKP under different conditions, allowing for the selection of a suitable buffer to maximize enzyme operational stability. FosAKP displays absolute specificity towards Fos; therefore, this enzyme was exploited for the development of an enzyme-based colorimetric biosensor. FosAKP was tethered at the bottom of a plastic cuvette using glutaraldehyde chemistry to develop a simple colorimetric method for the determination of Fos in drinking water and animal plasma.

Enhanced inhibition of human and rat aromatase activity by benzene ring substitutions in bisphenol A: QSAR structure-activity relationship and in silico docking analysis

Bisphenol A (BPA) is a widely used plastic material, but its potential endocrine disrupting effect has restricted its use. The BPA alternatives have raised concerns. This study aimed to compare inhibitory potencies of 11 BPA analogues on human and rat placental aromatase (CYP19A1). The inhibitory potency on human CYP19A1 ranged from bisphenol H (IC50, 0.93 μM) to tetramethyl BPA and tetrabromobisphenol S (ineffective at 100 μM) when compared to BPA (IC50, 73.48 μM). Most of them were mixed/competitive inhibitors and inhibited estradiol production in human BeWo cells. Molecular docking analysis showed all BPA analogues bind to steroid active site or in between steroid and heme of CYP19A1 and form a hydrogen bond with catalytic residue Met374. Pharmacophore analysis showed that there were 4 hydrophobic regions for BPA analogues, with bisphenol H occupying 4 regions. Bivariate correlation analysis showed that LogP (lipophilicity) and LogS (water solubility) of BPA analogues were correlated with their IC50 values. Computerized drug metabolism and pharmacokinetics analysis showed that bisphenol H, tetrabromobisphenol A, and tetrachlorobisphenol A had low solubility, which might explain their weaker inhibition on estradiol production on BeWo cells. In conclusion, BPA analogues mostly can inhibit CYP19A1 and the lipophilicity determines their inhibitory strength.

Long-Term Heavy Metal Pollution Induces Complex Differences in Farmland Topsoil and Rhizosphere Microbial Communities

The microbial effect of long-term heavy metal pollution on farmland remains unclear. Here, we investigated microbial (bacterial and fungal) communities in topsoil and rhizosphere samples with heavy metal (Cd, Cu, Pb, and Zn) pollution from four different types of tillage plots around an abandoned zinc smelter set up 85 years ago and analyzed the complex relationship between microorganisms, plants, and heavy metals (HMs) in soil to guide strategies for further soil remediation measures. The abundance and diversity index results showed that the bacterial and fungal diversities of the four plots were significantly different. Meanwhile, correlation analysis of the microbial communities and HMs showed that bacteria Pseudomonas and fungi Chaetothyriales and Fusarium had a good tolerance for HM pollution, but bacteria Vicinamibacteraceae, JG30_KF_CM45, RB41, Gaiella, MB-A2-108, 67-14, and Microvirga, and fungi Glomerellales, Hypocreales, Chaetomium, and Mortierella all showed indications of being sensitive to HM toxicity. Our structural equation model (SEM) attributed the inhibition of Zn and the promotion of Cd to bacterial diversity, attributed a weak inhibition of Cd to fungal diversity, and revealed the effects of the tillage type on these diversities.

Impact of potato starch on the inhibition of pancreatic lipase by potato phenolic acids

Phenolic acids are known to bind with and inhibit digestive enzymes in vitro. In particular, their action on pancreatic lipase at physiological pH is not well understood. Therefore, this study evaluated the inhibitory potential of phenolic acids from potato (caffeic acid CA, chlorogenic acid ChA, p-coumaric acid pCA, ferulic acid FA) towards lipase. All the phenolic acids were weak inhibitors of lipase and ChA was the weakest of all. Binding energy between phenolic acids and lipase obtained by molecular docking and solubility of the phenolic acids seemed to correlate well with inhibition data obtained at pH 6.9. Additionally, phenolic acids can form complexes with polysaccharides. In food such as potato, the presence of significant amount starch could therefore prevent phenolic acids to modulate pancreatic lipase. Consequently, we evaluated the effect of potato starch (up to 1 % w/w) on the ability to inhibit the activity of pancreatic lipase. The results showed that the presence of starch had essentially no effect on the inhibition of lipase by FA and pCA, that it led to decreased CA's inhibitory activity and to increased ChA's inhibitory activity. These contradictory observations could be explained by starch-phenolic acid complexation although they may be modulated by physical effects due to starch (microphase separation, decreased diffusion), and potentially amplified by the limited solubility of the phenolic acids.

Novel Small Molecule VLA-4 Inhibitors Optimized for Extended Hematopoietic and Progenitor Cell Mobilization

Background: Hematopoietic stem and progenitor cell (HSPC) transplant is an essential treatment for a variety of blood disorders and malignancies, and more recently, for gene editing therapies such as sickle cell anemia. A key step in this procedure is the effective mobilization of a large quantity of primitive donor HSPCs to ensure a durable and long lasting engraftment capable of developing into all blood cell lineages. The most commonly used regimen for donor mobilization is a 5-day course of Granulocyte Colony Stimulating Factor (G-CSF). However, G-CSF does not effectively mobilize a sufficient number or quality of stem cells in all donors, can have unwanted side effects, and is contraindicated in sickle cell disease. Therefore, it is desirable to identify an alternative treatment that is safe, rapid and cost effective. The cell surface integrin receptor VLA-4 (a4b1) and the chemokine receptor CXCR4 are essential for HSPC homing and retention in the bone marrow. Inhibition of these receptors rapidly mobilizes HSPCs within an hour, as opposed to the multi-day process using G-CSF. Small molecule inhibitors of VLA-4 have been reported previously, but most have poor pharmacokinetic properties and/or weak inhibition of VLA4. These inhibitors rapidly mobilize HSPCs into the peripheral blood of mice after a single s.c. injection, but their effect is short lived, yielding an inadequate number of HSPCs necessary for stem cell transplantation or for gene therapy. Therefore, VLA4 inhibitors with improved pharmacokinetic properties imparting extended and greater HSPC mobilization after a single dose is desirable. Methods: We designed and synthesized multiple novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. Inhibitors determined to be most potent with improved aqueous solubility were then tested in vivo in DBA mice for extended HSPC mobilization beyond 4 hours after a single SC injection, alone and in combination with the CXCR4 inhibitors. Results: We developed two main core molecules that each incorporate key VLA4 receptor binding motifs critical for potent inhibition of VLA4 binding to VCAM-1. Our first generation of inhibitors were quite potent and rapidly mobilized HSPCs, but their effect was short lived and yielded insufficient numbers of HSPCs for gene editing and engraftment studies. Most had poor solubility and very short plasma half-lives after s.c. injection in mice. We designed later generation analogues that strategically exploited a region of each optimized core, allowing for covalent addition of polyethylene glycol (PEG) chains of increasing length without decreasing potency. We determined that a minimum PEG length of 24 PEG units was required for extended HSPC mobilization in mice. WU-106, one of our VLA-4 inhibitors containing 24 PEG units, exhibited excellent aqueous solubility, longer plasma half-life after s.c. dosing, and mobilized 2-fold more murine HSPCs for a longer time than previously published best-in-class VLA-4 inhibitors or our first generation VLA4 inhibitor leads. Importantly, a synergistic effect on HSPC mobilization was achieved when WU-106 was co-administered with CXCR4 inhibitors (Plerixafor or BL-8040). Further optimization by the addition of longer PEG chains, even up to 40KD, retained sub nM potency and extended mobilization beyond 24 hours after a single injection. Our 20kD PEG analogues attached to our two different core molecules (WU-125 and WU-133) allowed for dosing of the drug in the evening, with maximum mobilization still present the next morning, thereby enabling HSPC collection to initiate soon (2-4 hours) after CXCR4 inhibitor dosing in the morning. The quantity of HSPCs obtained using this staggered dosing regimen was greater than simultaneous administration of the VLA-4 and CXCR4 inhibitors ( Fig. 1). Furthermore, our novel, long-acting PEG analogues demonstrated superior HSPC mobilization compared to an alpha 4-targeted antibody (anti-CD49d) in mice ( Fig.2). Conclusion: We have developed novel, potent PEGylated small molecule VLA-4 inhibitors that have excellent aqueous solubility and pharmacokinetics. The kinetics and magnitude of HSPC mobilization by our PEGylated inhibitors are superior to other previously described VLA-4 inhibitors and an antibody to alpha-4(anti-CD49d).

Identification of new potential candidates to inhibit EGF via machine learning algorithm

One of the cost-effective alternative methods to find new inhibitors has been the repositioning approach of existing drugs. The advantage of computational drug repositioning method is saving time and cost to remove the pre-clinical step and accelerate the drug discovery process. Hence, an ensemble computational-experimental approach, consisting of three steps, a machine learning model, simulation of drug-target interaction and experimental characterization, was developed. The machine learning type used here was a different tree classification method, which is one of the best randomize machine learning model to identify potential inhibitors from weak inhibitors. This model was trained more than one-hundred times, and forty top trained models were extracted for the drug repositioning step. The machine learning step aimed to discover the approved drugs with the highest possible success rate in the experimental step. Therefore, among all the identified molecules with more than 0.9 probability in more than 70% of the models, nine compounds, were selected. Besides, out of the nine chosen drugs, seven compounds have been confirmed to inhibit EGF in the published articles since 2019. Hence, two identified compounds, in addition to gefitinib, as a positive control, five weak-inhibitors and one neutral, were considered via molecular docking study. Finally, the eight proposed drugs, including gefitinib, were investigated using MTT assay and In-Cell ELISA to characterize the drugs' effect on A431 cell growth and EGF-signaling. From our experiments, we could conclude that salicylic acid and piperazine could play an EGF-inhibitor role like gefitinib.

Enzyme adaptation in Sphagnum peatlands questions the significance of dissolved organic matter in enzyme inhibition

Peatlands store a large proportion of global soil carbon in the form of peat because decomposition of plant organic matter is slow. In Sphagnum-dominated peatlands, dissolved organic matter (DOM) is traditionally considered an important inhibitor of hydrolytic enzymes due to the polyphenolic polymers it contains. Interestingly, the acidic character of the polymers in such DOM has never been tested for its enzyme-inhibitory properties. We raised two principal hypotheses: (1) not only the polyphenolic but also the acidic character of DOM inhibits the activity of extracellular enzymes in Sphagnum-dominated peatlands; (2) environmental, peat-extracted enzymes will show adaptation to their environment.We tested the inhibition of commercial acid phosphatase and cellobiohydrolase, and environmental phosphatase and β-glucosidase by following dissolved substances: (1) polyphenol-free polycarboxylates from Sphagnum cell walls, i.e. sphagnan, (2) environmental DOM (peat-DOM) containing polymers of polyphenolic–polycarboxylate nature, (3) tannic acid (carboxyl-free polyphenolic oligomer) and (4) monomeric phenolic acids.Sphagnan strongly inhibited commercial acid phosphatase, to a similar extent as peat-DOM and more strongly than tannic acid and a polycarboxylate from another moss (Leucobryum glaucum). Monomeric phenolic acids were weak inhibitors. Commercial cellobiohydrolase was only partially inhibited by sphagnan or peat-DOM. Environmental phosphatase and β-glucosidase were consistently slightly inhibited by tannic acid, but not by sphagnan or peat-DOM. Inhibition of commercial phosphatase by sphagnan and peat-DOM was counteracted by a polycation chitosan, indicating the electrostatic nature of carboxylate-mediated inhibition.Our results question the polyphenol-mediated enzyme inhibition in Sphagnum-dominated peatlands as (1) the DOM had a strong inhibitory potential due to its polycarboxylates; nevertheless, (2) the peat microbial communities exhibited enzyme resistance to both polyphenol and polycarboxylate polymers in peat-DOM.

A gastrointestinal locally activating Janus kinase inhibitor to treat ulcerative colitis

In this study, we integrated machine learning (ML), structure-tissue selectivity-activity-relationship (STAR), and wet lab synthesis/testing to design a gastrointestinal (GI) locally activating JAK inhibitor for ulcerative colitis treatment. The JAK inhibitor achieves site-specific efficacy through high local GI tissue selectivity while minimizing the requirement for JAK isoform specificity to reduce systemic toxicity. We used the ML model (CoGT) to classify whether the designed compounds were inhibitors or noninhibitors. Then we used the regression ML model (MTATFP) to predict their IC50 against related JAK isoforms of predicted JAK inhibitors. The ML model predicted MMT3-72, which was retained in the GI tract, to be a weak JAK1 inhibitor, while MMT3-72-M2, which accumulated in only GI tissues, was predicted to be an inhibitor of JAK1/2 and TYK2. ML docking methods were applied to simulate their docking poses in JAK isoforms. Application of these ML models enabled us to limit our synthetic efforts to MMT3-72 and MMT3-72-M2 for subsequent wet lab testing. The kinase assay confirmed MMT3-72 weakly inhibited JAK1, and MMT3-72-M2 inhibited JAK1/2 and TYK2. We found that MMT3-72 accumulated in the GI lumen, but not in GI tissue or plasma, but released MMT3-72-M2 accumulated in colon tissue with minimal exposure in the plasma. MMT3-72 achieved superior efficacy and reduced p-STAT3 in DSS-induced colitis. Overall, the integration of ML, the structure-tissue selectivity-activity-relationship system, and wet lab synthesis/testing could minimize the effort in the optimization of a JAK inhibitor to treat colitis. This site-specific inhibitor reduces systemic toxicity by minimizing the need for JAK isoform specificity.

Synthesis and Evaluation of Small Molecule Inhibitors of the Androgen Receptor N-Terminal Domain.

The androgen receptor (AR) is central to prostate cancer pathogenesis and has been extensively validated as a drug target. However, small-molecule anti-androgen therapies remain limited due to resistance and will eventually fail to suppress tumor growth, resulting in progression to castration-resistant prostate cancer (CRPC). The intrinsically disordered N-terminal domain (NTD) is crucial for AR transactivation and has been investigated as a suitable target in the presence of ligand binding domain mutations. A screening campaign identified biaryl isoxazole compound 7 as a weak inhibitor of the AR NTD. A library of biaryl analogues were synthesized, and their biological activities were assessed in a VCaP cell-based luciferase reporter gene assay. A structure-activity relationship (SAR) study revealed that indazole analogue 16 exhibited increased potency and favorable physicochemical properties with a benchmarked pharmacokinetic profile, providing a suitable starting point for further optimization of 16 as a CRPC therapeutic in the presence of AR mutations.

Population pharmacokinetics of mirvetuximab soravtansine in patients with folate receptor-α positive ovarian cancer: The antibody-drug conjugate, payload and metabolite.

Mirvetuximab soravtansine is a first-in-class antibody-drug conjugate recently approved for the treatment of folate receptor-α positive ovarian cancer. The aim of this study was to develop a population pharmacokinetic model to describe the concentration-time profiles of mirvetuximab soravtansine, the payload (DM4) and a metabolite (S-methyl-DM4). Mirvetuximab soravtansine was administered intravenously from 0.15 to 7mg/kg to 543 patients with predominantly platinum-resistant ovarian cancer in 3 clinical studies, and the plasma drug concentrations were analysed using a nonlinear mixed-effects modelling approach. Stepwise covariate modelling was performed to identify covariates. We developed a semi-mechanistic population pharmacokinetic model that included linear and nonlinear routes for the elimination of mirvetuximab soravtansine and a target compartment for the formation and disposition of the payload and metabolite in tumour cells. The clearance and volume of the central compartment were 0.0153 L/h and 2.63 L for mirvetuximab soravtansine, 8.83 L/h and 3.67 L for DM4, and 2.04 L/h and 6.3L for S-methyl-DM4, respectively. Body weight, serum albumin and age were identified as statistically significant covariates. Exposures in patients with renal or hepatic impairment and who used concomitant cytochrome P450 (CYP) 3A4 inhibitors were estimated. There is no need for dose adjustment due to covariate effects for mirvetuximab soravtansine administered at the recommended dose of 6mg/kg based on adjusted ideal body weight. Dose adjustment is not required for patients with mild or moderate renal impairment, mild hepatic impairment, or when concomitant weak and moderate CYP3A4 inhibitors are used.

Assessing the Potential of Gallic Acid and Methyl Gallate to Enhance the Efficacy of β-Lactam Antibiotics against Methicillin-Resistant Staphylococcus aureus by Targeting β-Lactamase: In Silico and In Vitro Studies.

Methicillin-resistant Staphylococcus aureus (MRSA), a global health concern, has prompted research into antibiotic adjuvants as a potential solution. Although our group previously reported the enhancing effects of gallic acid (GA) and methyl gallate (MG) on penicillin G activity against MRSA, the synergistic potential with other β-lactam antibiotics and the underlying mechanism have not been fully explored. Therefore, this study primarily aimed to investigate the antibacterial synergism with β-lactam antibiotics through disc diffusion, checkerboard, and time-kill assays. The β-lactamase inhibition was also examined through both molecular modeling and in vitro experiments. Additionally, bacterial morphology changes were studied using a scanning electron microscopy (SEM). The results revealed that both GA and MG exhibited anti-MRSA activity and showed indifferent effects when combined with β-lactam antibiotics against methicillin susceptible S. aureus (MSSA). Interestingly, MG demonstrated synergism with only the β-lactamase-unstable antibiotics against MRSA with the lowest fractional inhibitory concentration (FIC) indexes of ≤3.75. However, GA and MG exhibited weak β-lactamase inhibition. Furthermore, GA, MG, and the combination with ampicillin induced the morphological changes in MRSA, suggesting a possible mechanism affecting the cell membrane. These findings suggest that MG could potentially serve as an adjunct to β-lactam antibiotics to combat MRSA infections.

Characteristics of compounds with strong or weak nitrification inhibition in sewage.

To clarify the characteristics of compounds with strong or weak nitrification inhibition in sewage, 64 organic compounds including compounds registered in Pollutant Release and Transfer Register (PRTR) were evaluated in terms of their chemical structures and molecular weights. Nineteen compounds showed strong nitrification inhibition by testing with Nitrosomonas europaea. Compounds with thioamide structures had the lowest median value of EC50 (0.017mg/L), followed by those with alkyne structures (0.121mg/L), chlorophenol structures (0.300mg/L), and then azole structures (0.365mg/L). In contrast, 33 of the 64 compounds showed weak nitrification inhibition at a concentration of 10 mg/L, 27 of which were categorized into three main groups: long-chain alcohol structures, alkyne structures with a phenyl group, and aromatic structures. Most compounds with strong nitrification inhibition had a low molecular weight (MW) from 50 to 200. Meanwhile, the proportion of compounds with weak nitrification inhibition tended to be greater with increasing MW and such compounds were predominant at higher molecular weights above 300. The correlations of results derived from tests of nitrification inhibition based on ISO 9509 and N. europaea showed that 24 out of 30 compounds provided results that were highly correlated between these tests (R = 0.85), while 4 compounds with chlorophenol structures and 2 compounds with alkyne structures showed weaker inhibition rates in the ISO 9509 test than in the N. europaea test. Our results indicate that the magnitude of nitrification inhibition depends on MW in addition to the chemical structure, which is helpful in the search for the cause of nitrification inhibition in wastewater treatment plants.

Discovery of BCL-XL Heterobifunctional Degrader with Potentially Improved Therapeutic Window and Minimal Platelet Toxicity for Hematological Malignancies

Background In hematologic malignancies, apoptosis evasion promotes tumor formation/progression and is driven by the overexpression of anti-apoptotic proteins, including BCL-2 and BCL-XL. BCL-2-targeting small molecule inhibitor venetoclax won FDA approval for chronic lymphocytic leukemia and acute myeloid leukemia. Interests in developing BCL-XL-selective inhibitors arise as BCL-XL overexpression is implicated in venetoclax resistance. However, these efforts failed in clinical development due to dose-limiting thrombocytopenia, as platelets (PLT) also depend on BCL-XL for survival. To mitigate this on-target toxicity, a degrader strategy has been proposed, whereby BCL-XL protein can be selectively degraded by the von Hippel-Lindau protein (VHL) E3 ligase in tumor cells, but not in PLT which minimally express VHL. The first such BCL-XL degrader (DT2216) was developed and is now in early clinical trials. Despite negligible PLT toxicity in vitro, DT2216 was still toxic to PLT in vivo. We reasoned that since earlier BCL-XL degraders used strong BCL-XL inhibitor as warhead, e.g. the warhead for DT2216 is ABT263, they could readily bind to and inhibit BCL-XL in PLT causing thrombocytopenia. Warhead-containing metabolites could also form and behave as an inhibitor, exacerbating PLT toxicity in vivo. Utilizing our artificial intelligence-empowered neoDegrader platform, which accurately characterizes the landscape of the degrader-mediated, metastable interactions between E3 and its target protein, we were able to computationally design molecules that were relatively weak BCL-XL binders but could maintain the ternary complex at metastable states. Herein, we report the preclinical profile of the lead candidate NXD02. Methods We assessed the binding affinity of NXD02 to BCL-XL by surface plasmon resonance. We conducted flow cytometry to quantify NXD02-mediated BCL-XL protein degradation and apoptotic effects in MOLT4 cells. We also quantified the anti-proliferative activity of NXD02 by CellTiter-Glo assay. We measured protein level changes of apoptosis markers by western blot. We simultaneously determined plasma concentration of NXD02 and PLT at various time points following a single injection of NXD02 in rodents and monkeys. Finally, we evaluated the anti-tumor activity of NXD02 in an orthotopicMOLT4-luc mouse xenograft (CDX) model by bioluminescence imaging. Results The binding affinity of NXD02 to BCL-XL was > 40-fold weaker than that to DT2216 (K D 28.7 vs 0.6 nM). Despite this, NXD02 strongly degraded BCL-XL protein (DC 50 6.6 nM) with no obvious hook effect, which was more potent than DT2216 (DC 50 17.4 nM) in MOLT4 cells. At 100 nM, NXD02 markedly increased levels of active caspase 3 and cleaved PARP, which was consistent with evidence of massive apoptosis. This was translated to a potent anti-proliferative effect comparable to DT2216 (IC 50 27.8 vs 42.7 nM). NXD02 did not affect PLT viability up to a concentration of 10 μM in vitro. Notably, a single dose of NXD02 at 10 mg/kg i.v. achieved an exposure >4-fold than that of DT2216 (dose-normalized AUC 68.5 vs 15.3 μg*h/mL/(mg/kg)) in CD1 mice, while producing similar levels of peak PLT reduction (58% vs 65%) at ~24 h, which recovered by day 3. Comparable or less PLT reduction was also observed in rats (64% vs 66%) at 15 mg/kg and in cynomolgus monkeys (63% for NXD02 vs 87% for DT2216) at 3 mg/kg, all of which fully recovered by days 5-6, supporting the possibility for weekly dosing. Furthermore, NXD02 demonstrated stronger in vivo anti-tumor activity than DT2216 in a CDX model. Weekly i.v. dosing of NXD02 at 3 and 10 mg/kg achieved 80% and 100% tumor growth inhibition (TGI) by day 28 respectively, as compared to 36% TGI for DT2216 at 10 mg/kg. There was no significant body weight loss for all groups. Conclusions We have discovered a structurally-novel, VHL-mediated, heterobifunctional BCL-XL degrader NXD02 with “Weak Inhibitor, Strong Degrader” properties. NXD02 displayed, at the same dose, a much higher drug exposure, better anti-tumor activity and comparable platelet toxicity profile in vivo than the most advanced BCL-XL degrader DT2216. The results suggest that NXD02 may have a more favorable safety margin. In vitro toxicity profiling of NXD02 including Ames test and safety panel revealed no concerns so far. In vivo safety assessment is ongoing for NXD02 as a promising candidate for clinical development in liquid and potentially select solid tumors.

Revumenib Monotherapy in Patients with Relapsed/Refractory KMT2Ar Acute Leukemias: Efficacy and Safety Results from the Augment-101 Phase 1/2 Study

Background: Revumenib (SNDX-5613), a potent, selective small-molecule inhibitor of the menin-histone-lysine N-methyltransferase 2A (KMT2A) interaction, is being investigated in patients (pts) with relapsed/refractory (R/R) KMT2A-rearranged ( KMT2Ar) and nucleophosmin 1-mutated ( NPM1m) acute leukemias. An initial analysis of the phase 1 of AUGMENT-101 (NCT04065399) has been reported ( Nature. 2023;615:920-924). Here we report an update of >1 year additional experience in the phase 1 portion of the study, which has completed enrollment, with safety data from 131 treated pts and efficacy data in 80 pts with R/R KMT2Ar leukemia. Demographic data from pivotal phase 2 cohorts are included; safety and efficacy data of a preplannedanalysis will be reported at the meeting. Methods: In phase 1, pts aged ≥30 days with R/R acute leukemias were assigned to 1 of 6 dose-escalation arms designed to identify a recommended phase 2 dose (RP2D) for concomitant administration of weak, moderate, or strong cytochrome P450 3A4 inhibitor (CYP3A4i) azole antifungal or no CYP3A4i. Pts received revumenib every 12 hours (q12h) or 3 times a day at a flat dose (≥40 kg) or a body surface area-based dose (<40 kg). Safety was assessed in all pts who received ≥1 dose of revumenib; responses are reported for all treated pts with KMT2Ar. In pts who underwent hematopoietic stem cell transplant (HSCT), responses reported are those achieved before HSCT. The Phase 2 study was initiated following identification of an RP2D of 163 mg (95 mg/m 2 if <40 kg) q12h with a strong CYP3A4i in 28-day cycles until unacceptable toxicity, lack of at least morphological leukemia-free state (MLFS) by end of cycle 4, or progressive disease without clinical benefit as defined by the investigator. The phase 2 primary objectives are evaluation of safety and tolerability of revumenib and assessment of the complete remission (CR) + CR with partial hematologic recovery (CRh) rate. The safety and efficacy of revumenib at an RP2D is being explored in 3 expansion cohorts: Cohort 2A, KMT2Ar acute lymphoblastic leukemia (ALL)/mixed phenotype acute leukemia; Cohort 2B, KMT2Ar acute myeloid leukemia (AML); and Cohort 2C, NPM1m AML. A preplanned pooled analysis of adult and pediatric pts with KMT2Ar acute leukemia will be conducted when a subset of Cohorts 2A+2B pts have completed a minimum of 4 cycles or have discontinued therapy. Results: Phase 1: As of June 19, 2023, 80 pts with R/R KMT2Ar acute leukemia were treated with revumenib in the 6 dose-escalation arms (Table 1). In adults with AML (n=51), the largest KMT2Ar subgroup, CR+CRh was 28% and overall response rate (ORR) was 59%. Responses were consistent in ALL/other leukemia subtype (n=15), with 27% CR+CRh and 53% ORR. ORR (61%) was similar in pediatric pts (<18 years, n=18), with 22% proceeding to HSCT. Although similar percentages of adult and children proceeded to transplant, consistent with common clinical practice in pediatrics, 75% of children transplanted went on to HSCT prior to achieving a CR/CRh (2 MLFS, 1 CRp) versus 17% of adults. In the safety population (n=131), 24% of pts had a ≥Grade 3 treatment-related adverse event (TRAE), with Grade 3 QTc prolongation in 8% and differentiation syndrome in 2% (Table 1). QTc prolongation remained as the only dose-limiting toxicity; no patients experienced ventricular arrhythmias. In addition, based on updated phase 1 pharmacokinetics, clinical activity, and safety data, an RP2D of 276 mg q12h without a strong CYP3A4i was established. Phase 2: In a preliminary analysis of Cohorts 2A and 2B (June 19, 2023), 88 pts with KMT2Ar acute leukemia were enrolled and treated with revumenib at an RP2D (Table 2). Ages ranged from 1.3 to 75 years (22 aged <18 y; Table 2). Leukemia subtypes included ALL (15%), AML (81%), and other (3%). Pts were heavily pretreated, having received a median of 3 prior lines of therapy (range 1-12); 38% received ≥4 prior lines and 48% had prior HSCT. Conclusions: These phase 1 data continue to demonstrate deep responses to revumenib and a manageable safety profile in heavily pretreated patients with R/R KMT2Ar acute leukemia across ages and subtypes. The lack of targeted therapies approved for KMT2Ar leukemia indicate the need for further investigation of novel, potentially transformative therapies such as revumenib. Enrollment in the AUGMENT-101 trial is ongoing. Pivotal Phase 2 safety and efficacy data for the KMT2Ar population will be reported at the meeting.

Influence of alkali and alkali earth metals on pyrolysis of tobacco waste

The effect of alkali and alkali earth metals on pyrolysis behavior of tobacco was revealed in this paper. Four alkali and alkali earth metal ions (Na+, K+, Ca2+, and Mg2+) were used to impregnate acid-washed tobacco. The addition of alkali and alkali earth metal ions in acid-washed tobacco increases the relative content of ketones, acids, nitrogen containing compounds, and esters, while inhibit the formation of anhydro sugars and aldehydes. The generation of solid residues is enhanced by the presence of alkali and alkali earth metal ions and the increase of K+ concentration. The addition of 0.5 mmol K+/g can increase the yield of solid residues from 13.7 wt% to 20.2 wt%. The increase of K+ concentration promotes the conversion of anhydro sugars to ketones. The effect of hot water-wash, acid-wash, and acid-infuse, which can control the concentration of alkali and alkali earth metal ions in the tobacco, on the product distribution of tobacco pyrolysis were also discussed. Acid-wash method and hot water-wash method promote the production of carbohydrate derivatives and inhibit the generation of nitrogen-containing products. After nitric acid-wash and hot water-wash pretreatment, the relative content of nitrogen-containing products decreased from 52.1% to 6.2% and 4.3%, respectively. Compared with inorganic acid-wash pretreatment method, organic acid-wash pretreatment method has weaker inhibition on nitrogen-containing products. Organic acid-wash pretreatment method promoted the formation of aromatics and aldehydes. Prewash methods facilitate the thermal decomposition of tobacco. The results from this study suggests that feedstock pretreatment could be used as a strategy of controlling aroma compounds formation from tobacco.

The Pharmacokinetics of the Nonsteroidal Mineralocorticoid Receptor Antagonist Finerenone.

Finerenone, a selective and nonsteroidal antagonist of the mineralocorticoid receptor, has received regulatory approval with the indication of cardiorenal protection in patients with chronic kidney disease associated with type 2 diabetes. It is rapidly and completely absorbed and undergoes first-pass metabolism in the gut wall and liver resulting in a bioavailability of 43.5%. Finerenone can be taken with or without food. The pharmacokinetics of finerenone are linear and its half-life is 2 to 3 h in the dose range of up to 20 mg. Cytochrome P450 (CYP) 3A4 (90%) and CYP2C8 (10%) are involved in the extensive biotransformation of finerenone to pharmacologically inactive metabolites, which are excreted via both renal (80%) and biliary (20%) routes. Moderate or severe renal impairment, or moderate hepatic impairment result in area-under-the-curve increases of finerenone (< 40%), which do not require a dose adjustment per se, as the starting dose is based on estimated glomerular filtration rate (eGFR) and titrated according to serum potassium levels and eGFR decline. No relevant effects of age, sex, body size or ethnicity on systemic finerenone exposure were identified. Modulators of CYP3A4 activity were found to affect finerenone exposure, consistent with its classification as a sensitive CYP3A4 substrate. Serum potassium should be monitored during drug initiation or dosage adjustment of either a moderate or weak CYP3A4 inhibitor or finerenone, and the dose of finerenone should be adjusted as appropriate. Its use with strong inhibitors is contraindicated and strong or moderate inducers of CYP3A4 should be avoided. Finerenone has no potential to affect relevant CYP enzymes and drug transporters.

Utilizing H2O2 for the selective flotation separation of chalcopyrite and galena with their surface pre-adsorbed with Isopropyl Ethyl Thiocarbamate

Chalcopyrite and galena often coexisted in ore deposits, and they are usually processed by bulk flotation and then separated to obtain valuable mixed concentrateds. In this paper, we propose a flotation reagent strategy, that uses Isopropyl Ethyl Thiocarbamate (IPETC) and hydrogen peroxide (H2O2) to achieve different floatability on chalcopyrite and galena, it is evident that chalcopyrite and galena with IPETC adsorbed on the surface can be separated by H2O2. The flotation results show that H2O2 has a great inhibition effect on galena and exhibits a weak inhibition effect on chalcopyrite. Mixture of chalcopyrite and galena with IPETC adsorbed on the surface was successfully separated by flotation with 15 mg·L-1 H2O2 at pH 7. The inhibition mechanism of H2O2 was analyzed by contact angle measurements, single-channel scanning Inductively Coupled Plasma emission spectroscopy (single-channel ICP) analysis, Fourier Transform Infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The characterization analysis indicates that the rate of H2O2 oxidation of galena is faster, resulting in more hydrophilic oxidation products such as oxides and hydroxides on the surface of galena, while the surface of chalcopyrite is less affected by H2O2. Also, compared with chalcopyrite, the adsorbed IPETC on the galena surface can be easily blanketed by oxidation products, so realizes the effective separation of chalcopyrite and galena.