Inhibition Profile Research Articles

P05 The role of JAK inhibitors in difficult-to-treat adult-onset Still’s disease: a case series

Abstract Introduction Adult-onset Still’s Disease (AOSD) is a rare systemic auto-inflammatory disorder characterised by fever, arthritis and rash. Some patients with AOSD exhibit resistance or intolerance to conventional treatments, necessitating alternative therapeutic strategies. This case series aims to explore the efficacy and safety of JAK inhibitors in managing difficult-to-treat AOSD. The driving force behind Still’s disease lies in the excessive and inappropriate production of pro-inflammatory cytokines, notably IL-6 and interferon. Given the pivotal role of JAK inhibitors in mitigating these cytokine pathways, this study highlights their potential therapeutic impact in the management of difficult to treat AOSD. Case description In this retrospective case series, three adult patients with difficult-to-treat AOSD were evaluated for their response to JAK inhibitors, specifically tofacitinib and baricitinib. Case 1: A 32-year-old male presented with a three-week history of fevers, night sweats, weight loss, and lymphadenopathy. Diagnosed with AOSD 18 months later, his treatment began with prednisolone and methotrexate, which was subsequently switched to azathioprine. Tocilizumab was then introduced, alleviating joint pain but causing dermatitis. He was switched to anakinra, allowing a reduction in prednisolone but failing to induce remission and resulting in abnormal liver functions and increased hepatitis B virus titers. Finally, he commenced tofacitinib, achieving symptom resolution, reduced serum ferritin levels, and cessation of steroids. Case 2: A 32-year-old woman presented with a six-month history of joint pain, swelling, stiffness, night sweats, weight loss, and a recurrent rash. Laboratory tests showed anemia, lymphopenia, elevated inflammatory markers, and high ferritin levels. Initial treatment with 60 mg oral prednisolone showed some response, but polyarthritis necessitated further intervention. Anakinra initially managed systemic symptoms, though joint swelling persisted. Tocilizumab was tried but proved ineffective. Subsequently, transitioning to baricitinib at 4 mg daily resulted in good symptomatic control. Case 3: An 18-year-old female presented with recurrent fevers, an intermittent macular rash, joint pains, diarrhoea and splenomegaly. Despite initial antibiotic treatment, persistent symptoms led to an AOSD diagnosis, prompting steroid therapy. Methotrexate failed to control symptoms and caused significant nausea, leading to a switch to tocilizumab. While anakinra initially resolved skin and joint symptoms, recurrent flares necessitated further changes. Azathioprine was trialed but discontinued due to side effects. Colchicine was tried next. Finally, tofacitinib was introduced, and one year later, she achieved significant progress, reducing prednisolone dosage to 5 mg. Discussion The findings from this case series underscore the potential of JAK inhibitors, particularly tofacitinib and baricitinib, as effective treatments for refractory AOSD. In all three cases, patients showed substantial clinical improvement and were able to reduce or discontinue steroids. This suggests a promising role for JAK inhibitors in managing AOSD, especially given the disease’s association with excessive pro-inflammatory cytokine production, including IL-6 and IFN pathways. The transition to JAK inhibitors was often necessitated by inadequate responses or adverse effects from conventional therapies like methotrexate, azathioprine, and biologics such as tocilizumab and anakinra. The successful outcomes with JAK inhibitors highlight their ability to achieve disease control where other treatments failed, demonstrating their efficacy in reducing systemic inflammation and disease activity. Further research is warranted to establish optimal dosing, duration of therapy, and long-term safety profiles of JAK inhibitors in this patient population. These preliminary results are encouraging and suggest a new avenue for treating AOSD. Key learning points • Efficacy of JAK inhibitors: JAK inhibitors, particularly tofacitinib and baricitinib, demonstrated substantial clinical improvement in patients with refractory AOSD. • Steroid reduction: All three patients were able to reduce or discontinue steroid use, highlighting the potential of JAK inhibitors to minimise long-term steroid dependency. • Management of refractory AOSD: JAK inhibitors provided effective disease control where conventional therapies such as methotrexate, azathioprine, and biologics like tocilizumab and anakinra were inadequate or resulted in adverse effects. • Mechanism of action: The efficacy of JAK inhibitors may be attributed to their ability to block pro-inflammatory cytokine pathways, particularly IL-6 and IFN, which are crucial in AOSD pathogenesis. • Safety profile: The treatment was well-tolerated, with significant symptomatic relief and no severe adverse events reported during the study period. • Need for further research: There is a need for further prospective studies to determine the optimal dosing, duration of therapy, and long-term safety profiles of JAK inhibitors in AOSD patients. • Personalised treatment approach: The study supports a personalised treatment approach, considering JAK inhibitors for patients with difficult-to-treat AOSD who do not respond adequately to conventional therapies.

Novel benzenesulfonamide-aroylhydrazone conjugates as carbonic anhydrase inhibitors that induce MAPK/ERK-mediated cell cycle arrest and mitochondrial-associated apoptosis in MCF-7 breast cancer cells

A series of novel 4-alkylthio-2-chloro-5-[(2-arylmethylidene)hydrazinecarbonyl]benzenesulfonamide derivatives 3–22 were synthesized and evaluated for their inhibitory activity against human carbonic anhydrase isozymes hCA I, hCA II, hCA IX, and hCA XII. These compounds showed varying degrees of activity against the studied isoenzymes. However, the importance of substituent choice in designing potent carbonic anhydrase inhibitors is highlighted by the strong inhibition profiles of compounds 3 and 10 against hCA IX and the low average KI values for compounds 9 and 10 (134 nM and 77 nM, respectively). All the synthesized compounds were evaluated for their antiproliferative activity toward HeLa, HCT-116, and MCF-7 cell lines. Compounds 9 and 19 exhibited significant activity, particularly against the MCF-7 cell line (IC50 values of 4 μM and 6 μM, respectively). Notably, compound 9 demonstrated a high selectivity index (SI = 8.2) for MCF-7 cells. The antiproliferative effects of compounds 9 and 19 were linked to the induction of cell cycle arrest and apoptosis via the mitochondrial pathway and involved the activation of the MAPK/ERK signaling pathway. Inhibition of MAPK/ERK activity reduced the compounds’ ability to induce cell cycle arrest and apoptosis, indicating the critical role of this pathway. These findings suggest that compounds 9 and 19 are promising candidates for further development as specific and potent anticancer agents targeting the MAPK/ERK pathway.

Establishment of a diverse pheno-genotypic challenge set of Klebsiella pneumoniae and Pseudomonas aeruginosa suitable for use in the murine pneumonia model.

Preclinical murine infection models lack inter-laboratory uniformity, complicating result comparisons and data reproducibility. The European Innovative Medicines initiative-funded consortium (COMBINE) has developed a standardized murine neutropenic pneumonia protocol to address these concerns. While model methods have been standardized, a major obstacle to consistent results is the lack of available bacteria with defined viability and variability. Herein, we establish a diverse challenge set of Klebsiella pneumoniae and Pseudomonas aeruginosa suitable for use in the COMBINE protocol to further minimize experimental inconsistency and improve the interpretability of data generated among differing laboratories. Sixty-six K. pneumoniae and 65 P. aeruginosa were phenotypically profiled against tigecycline (K. pneumoniae only), levofloxacin, meropenem, cefiderocol and tobramycin. Fifty-nine isolates were introduced into the COMBINE model to assess the sufficiency of the starting bacterial inoculation, resultant baseline bacterial burden, achievement of ≥1 log10cfu/lung growth at 24 h, time to and percentage mortality. Forty-five isolates displaying desirable minimum inhibitory concentration profiles were subjected to replicate in vivo testing to assess target parameters. 83% of K. pneumoniae reached the prerequisite growth at 24 h using a starting bacterial burden ≥7 log10cfu/lung. P. aeruginosa isolates grew well in the model: 90% achieved the growth target with a starting bacterial burden of 6 log10cfu/lung. Mortality was negligible for K. pneumoniae but high for P. aeruginosa. Poor or inconsistent achievement of the 24 h growth target was seen in 11/59 isolates. With this diverse cache of viable isolates established in the COMBINE pneumonia model, future translational studies can be undertaken to set efficacy benchmarks among laboratories.

Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Synthesis, Characterizations, Anti-Diabetic and Molecular Modeling Approaches of Hybrid Indole-Oxadiazole Linked Thiazolidinone Derivatives

Objective: To synthesize hybrid compounds of indole and oxadiazole in search of highly effective anti-diabetic therapeutic agent. Methods: With the goal of advancing diabetes research, our group designed and synthesized a library of 15 compounds based on indole-derived oxadiazole bearing varied substituted thiazolidinone via a multistep synthetic route. 13C-NMR, 1H-NMR and HREI-MS were applied for the characterization of all the synthesized compounds. Their biological inhibitory activity against diabetic enzymes, i.e., α-amylase and α-glucosidase was also determined. Results: Compound 7, 9 and 15 exhibited excellent inhibition against α-amylase and α-glucosidase than the standard acarbose (IC50 = 8.50 ± 0.10 µM for α-amylase and 9.30 ± 0.30 µM for α-glucosidase. To ensure the inhibitory actions of these potent analogs in molecular docking, an in silico approach was used. To determine the drug likeness of the reported analogs, an ADMET investigation was also carried out to explore the nature of the designed compounds if used as a drug. Conclusion: Fluoro-substituted analog 15 has stronger inhibition profile against both enzymes. All the potent compounds can be used as effective anti-diabetic therapeutic agents in future.

Unrivalled Insight into Possible Biopharmaceutical Application of Justicia vahlii Roth. (Acanthaceae): Chemodiversity, In Vitro Bioactivities, and Computational Analysis.

Justicia vahliiRoth. is an important wild medicinal food plant traditionally used for treating inflammation and various common ailments. This study investigated the chemical composition, antioxidant, enzyme inhibition and toxicity profiles of n-hexane (nHEJv) and chloroform (CEJv) extracts of J. vahlii. Moreover, the effect of the extracts was evaluated on CCl4 induced liver injury. The total phenolic and flavonoid contents were present in both extracts in significant amount. The UPLC-Q-TOF-MS and GC-MS profiling of CEJv tentatively identified several important phytocompounds. The CEJv extract was comparatively more active for antioxidant activity and α-amylase inhibition, whereas the nHEJv extract presented higher inhibition potential against urease, tyrosinase, and α-glucosidase enzymes. Similarly, the in-silicostudy of four major compounds, i. e., 1-acetoxypinoresinol, 3-hydroxysebacic acid, nortrachelogenin, and viscidulin-III have shown a good docking score against the clinically significant enzymes. The acute oral toxicity and brine shrimp lethality assaysrevealed the extracts as non-toxic. The CCl4 treated animals showed a geared depletion of various antioxidant enzymes which were significantly reversed with the treatment of the extracts. Overall, the study's findings revealed J. vahliiwith antioxidant mediated hepatoprotective and enzyme inhibition potential and warrant further research on isolation of the bioactive compounds.

Discovery of ICOS-Targeted Small Molecules Using Affinity Selection Mass Spectrometry Screening.

Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules. To the best of our knowledge, this is the first study that utilizes ASMS screening to discover small molecules targeting immune checkpoints. Compound 9 with a promising ICOS/ICOS-L inhibitory profile (IC50=29.38±3.41 μM) was selected as the template for the modification. Following preliminary structure-activity relationship (SAR) study and molecular dynamic (MD) simulation revealed the critical role of the ortho-hydroxy group on compound 9 in the ICOS binding, as it could stabilize the interaction via the hydrogen bond formation with residuals on the glycan, and the depletion could lead to an activity lost. This work validates a promising inhibitor for the ICOS/ICOS-L interaction, and we anticipate future modifications could provide more potent modulators for this interaction.

First-in-human dose escalation study of the first-in-class PDE3A-SLFN12 complex inducer BAY 2666605 in patients with advanced solid tumors co-expressing SLFN12 and PDE3A.

To evaluate the safety, tolerability, and pharmacokinetics of BAY 2666605, a velcrin that induces complex formation between the phosphodiesterase PDE3A and the protein Schlafen 12 (SLFN12) leading to a cytotoxic response in cancer cells. This was a first-in-human phase I study of BAY 2666605 (NCT04809805), an oral, potent first-in-class PDE3A-SLFN12 complex inducer, with reduced PDE3A inhibition. Adults with advanced solid tumors that co-express SLFN12 and PDE3A received BAY 2666605 at escalating doses starting at 5 mg once daily in 28-day cycles. Forty-seven patients were pre-screened for SLFN12 and PDE3A overexpression, and 5 biomarker-positive patients received ≥ 1 BAY 2666605 dose. The most common adverse event was grade 3-4 thrombocytopenia in 3 of the 5 patients treated. The long half-life (> 360 hours) and associated accumulation of BAY 2666605 led to the selection of an alternative schedule consisting of a loading dose with QD maintenance dose. The maximum tolerated dose was not established as the highest doses of both schedules were intolerable. No objective responses were observed. Due to the high expression of PDE3A in platelets compared to tumor tissues, the ex vivo dose-dependent inhibitory effect of BAY 2666605 on megakaryocytes, and the pharmacokinetic profile of the compound, alternative schedules were not predicted to ameliorate the mechanism-based thrombocytopenia. Despite the decreased PDE3A enzymatic inhibition profile of BAY 2666605, the occurrence of thrombocytopenia in treated patients, an on-target effect of the compound, precluded the achievement of a therapeutic window, consequently leading to trial termination.

"Pseudosubstrate Envelope"/Free Energy Perturbation-Guided Design and Mechanistic Investigations of Benzothiazole HIV Capsid Modulators with High Ligand Efficiency.

Based on our proposed "pseudosubstrate envelope" concept, 25 benzothiazole-bearing HIV capsid protein (CA) modulators were designed and synthesized under the guidance of free energy perturbation technology. The most potent compound, IC-1k, exhibited an EC50 of 2.69 nM against HIV-1, being 393 times more potent than the positive control PF74. Notably, IC-1k emerged as the highest ligand efficiency (LE = 0.32) HIV CA modulator, surpassing that of the approved drug lenacapavir (LE = 0.21). Surface plasmon resonance assay and crystallographic analysis confirmed that IC-1k targeted HIV-1 CA within the chemical space of the "pseudosubstrate envelope". Further mechanistic studies revealed a dual-stage inhibition profile: IC-1k disrupted early-stage capsid-host-factor interactions and promoted late-stage capsid misassembly. Preliminary pharmacokinetic evaluations demonstrated significantly improved metabolic stability in human liver microsomes for IC-1k (T1/2 = 91.3 min) compared to PF74 (T1/2 = 0.7 min), alongside a favorable safety profile. Overall, IC-1k presents a promising lead compound for further optimization.

Activity of novel virus families infecting soil nitrifiers is concomitant with host niche differentiation.

Chemolithoautotrophic nitrifiers are model groups for linking phylogeny, evolution, and ecophysiology. Ammonia-oxidising bacteria (AOB) typically dominate the first step of ammonia oxidation at high ammonium supply rates, ammonia-oxidising archaea (AOA) and complete ammonia-oxidising Nitrospira (comammox) are often active at lower supply rates or during AOB inactivity, and nitrite-oxidising bacteria (NOB) complete canonical nitrification. Soil virus communities are dynamic but contributions to functional processes are largely undetermined. In addition, characterising viruses infecting hosts with low relative abundance, such as nitrifiers, may be constrained by vast viral diversity, partial genome recovery, and difficulties in host linkage. Here, we describe a targeted incubation study that aimed to determine whether growth of different nitrifier groups in soil is associated with active virus populations and if process-focussed analyses facilitate characterisation of high-quality virus genomes. dsDNA viruses infecting different nitrifier groups were enriched in situ via differential host inhibition. Growth of each nitrifier group was consistent with predicted inhibition profiles and concomitant with the abundance of their viruses. These included 61 high-quality/complete virus genomes 35-173kb in length with minimal similarity to validated families. AOA viruses lacked ammonia monooxygenase sub-unit C (amoC) genes found in marine AOA viruses but some encoded AOA-specific multicopper oxidase type 1 (MCO1), previously implicated in copper acquisition, and suggesting a role in supporting energy metabolism of soil AOA. Findings demonstrate focussed incubation studies facilitate characterisation of active host-virus interactions associated with specific processes and viruses of soil AOA, AOB and NOB are dynamic and potentially influence nitrogen cycling processes.

In vitro Cyclooxygenase inhibitory activity and GC-MS profiling of bioactive compounds in Bauhinia racemosa Lam.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a common treatment for chronic pain and inflammation. While NSAIDs have been shown to reduce inflammation and pain associated with them, they have an array of side effects. Researchers investigated the COX inhibitory action of NSAIDs. They revealed that there are two distinct COX enzymes: COX-1 and COX2. Developing COX inhibitors has remained critical for producing innovative and safe anti-inflammatory drugs. Bauhinia racemosa Lam belonging to the family Fabaceae has a wide range of medicinal properties. The plant is used to treat various diseases in traditional medicine, but there is less knowledge based on clinical efficacy as an anti-inflammatory agent. In the present study Bauhinia racemosa Lam hydroalcoholic extract were screened for COX inhibition and GC-MS analysis was done to screen phytochemicals. The results obtained were found to inhibit 89% of COX activity significantly as compared to standard drug (Diclofenac sodium). GC-MS analysis revealed a total of ten potent bioactive compounds. The samples were identified by comparing retention time and peak area to literature and interpreting mass spectra. Results observed from the study suggest that the hydroalcoholic extract of Bauhinia racemosa Lam possess anti-inflammatory activity and can be utilized as an alternate source for NSAIDs. Keywords: COX, Bauhinia racemosa Lam, Anti-inflammatory, NSAIDs.

Efficacy of Integrase Strand Transfer Inhibitors and the Capsid Inhibitor Lenacapavir against HIV-2, and Exploring the Effect of Raltegravir on the Activity of SARS-CoV-2

Retroviruses perpetuate their survival by incorporating a copy of their genome into the host cell, a critical step catalyzed by the virally encoded integrase. The viral capsid plays an important role during the viral life cycle, including nuclear importation in the case of lentiviruses and integration targeting events; hence, targeting the integrase and the viral capsid is a favorable therapeutic strategy. While integrase strand transfer inhibitors (INSTIs) are recommended as first-line regimens given their high efficacy and tolerability, lenacapavir is the first capsid inhibitor and the newest addition to the HIV treatment arsenal. These inhibitors are however designed for treatment of HIV-1 infection, and their efficacy against HIV-2 remains widely understudied and inconclusive, supported only by a few limited phenotypic susceptibility studies. We therefore carried out inhibition profiling of a panel of second-generation INSTIs and lenacapavir against HIV-2 in cell culture, utilizing pseudovirion inhibition profiling assays. Our results show that the tested INSTIs and lenacapavir exerted excellent efficacy against ROD-based HIV-2 integrase. We further evaluated the efficacy of raltegravir and other INSTIs against different variants of SARS-CoV-2; however, contrary to previous in silico findings, the inhibitors did not demonstrate significant antiviral activity.

Improving Predictive Efficacy for Drug Resistance in Novel HIV-1 Protease Inhibitors through Transfer Learning Mechanisms.

The human immunodeficiency virus presents a significant global health challenge due to its rapid mutation and the development of resistance mechanisms against antiretroviral drugs. Recent studies demonstrate the impressive performance of machine learning (ML) and deep learning (DL) models in predicting the drug resistance profile of specific FDA-approved inhibitors. However, generalizing ML and DL models to learn not only from isolates but also from inhibitor representations remains challenging for HIV-1 infection. We propose a novel drug-isolate-fold change (DIF) model framework that aims to predict drug resistance score directly from the protein sequence and inhibitor representation. Various ML and DL models, inhibitor representations, and protein representations were analyzed through realistic validation mechanisms. To enhance the molecular learning capacity of DIF models, we employ a transfer learning approach by pretraining a graph neural network (GNN) model for activity prediction on a data set of 4855 HIV-1 protease inhibitors (PIs). By performing various realistic validation strategies on internal and external genotype-phenotype data sets, we statistically show that the learned representations of inhibitors improve the predictive ability of DIF-based ML and DL models. We achieved an accuracy of 0.802, AUROC of 0.874, and r of 0.727 for the unseen external PIs. By comparing the DIF-based models with a null model consisting of isolate-fold change (IF) architecture, it is observed that the DIF models significantly benefit from molecular representations. Combined results from various testing strategies and statistical tests confirm the effectiveness of DIF models in testing novel PIs for drug resistance in the presence of an isolate.

Optimization of Screening Media to Improve Antimicrobial Biodiscovery from Soils in Undergraduate/Citizen Science Research-Engaged Initiatives.

Background/Objectives: Research-engaged academic institutions offer the opportunity to couple undergraduate education/citizen science projects with antimicrobial biodiscovery research. Several initiatives reflecting this ethos have been reported internationally (e.g., Small World, Tiny Earth, MicroMundo, Antibiotics Unearthed). These programs target soil habitats due to their high microbial diversity and promote initial screening with non-selective, nutrient media such as tryptic soy agar (TSA). However, evaluation of published outputs to date indicates that isolate recovery on TSA is consistently dominated by the genera Bacillus, Pseudomonas, and Paenibacillus. In this study, we evaluated the potential of soil extract agar to enhance soil isolate diversity and antibiosis induction outcomes in our undergraduate Antibiotics Unearthed research program. Methods: We comparatively screened 229 isolates from woodland and garden soil samples on both tryptic soy agar (TSA) and soil extract agar (SEA) for antimicrobial activity against a panel of clinically relevant microbial pathogens. Results: On one or both media, 15 isolates were found to produce zones of clearing against respective pathogens. 16S rRNA gene sequencing linked the isolates with three genera: Streptomyces (7), Paenibacillus (6), and Pseudomonas (2). Six of the Streptomyces isolates and one Pseudomonas demonstrated antimicrobial activity when screened on SEA, with no activity on TSA. Furthermore, incorporation of the known secondary metabolite inducer N acetyl-glucosamine (20 mM) into SEA media altered the pathogen inhibition profiles of 14 isolates and resulted in broad-spectrum activity of one Streptomyces isolate, not observed on SEA alone. In conclusion, SEA was found to expand the diversity of culturable isolates from soil and specifically enhanced the recovery of members of the genus Streptomyces. SEA was also found to be a superior media for antibiosis induction among Streptomyces isolates when compared to TSA. It was noted that Paenibacillus isolates' antibiosis induction demonstrated a strain-specific response with respect to the growth media used. Conclusions: The authors propose SEA inclusion of in soil screening protocols as a cost-effective, complementary strategy to greatly enhance outcomes in undergraduate/citizen science-engaged antimicrobial biodiscovery initiatives.

Targeting Hypoglycemic Natural Products from the Cloud Forest Plants Using Chemotaxonomic Computer-Assisted Selection.

The cloud forest (CF), a hugely biodiverse ecosystem, is a hotspot of unexplored plants with potential for discovering pharmacologically active compounds. Without sufficient ethnopharmacological information, developing strategies for rationally selecting plants for experimental studies is crucial. With this goal, a CF metabolites library was created, and a ligand-based virtual screening was conducted to identify molecules with potential hypoglycemic activity. From the most promising botanical families, plants were collected, methanolic extracts were prepared, and hypoglycemic activity was evaluated through in vitro enzyme inhibition assays on α-amylase, α-glucosidase, and dipeptidyl peptidase IV (DPP-IV). Metabolomic analyses were performed to identify the dominant metabolites in the species with the best inhibitory activity profile, and their affinity for the molecular targets was evaluated using ensemble molecular docking. This strategy led to the identification of twelve plants (in four botanical families) with hypoglycemic activity. Sida rhombifolia (Malvaceae) stood out for its DPP-IV selective inhibition versus S. glabra. A comparison of chemical profiles led to the annotation of twenty-seven metabolites over-accumulated in S. rhombifolia compared to S. glabra, among which acanthoside D and cis-tiliroside were noteworthy for their potential selective inhibition due to their specific intermolecular interactions with relevant amino acids of DPP-IV. The workflow used in this study presents a novel targeting strategy for identifying novel bioactive natural sources, which can complement the conventional selection criteria used in Natural Product Chemistry.

Investigating the efficacy of naphthalene-thiazole hybrid hydrazones as α-glucosidase inhibitors

In this study, a series of hydrazone derivatives containing naphthalene and thiazole hybrids were synthesized through the condensation of (E)-4-methyl-2-(2-(naphthalen-1-ylmethylene)hydrazineyl) thiazole-5-carbohydrazide with various carbonyl compounds, including aldehydes and ketones, whether carbocyclic aromatic or heterocyclic, using a grinding method with drops of acetic acid. The elucidation of the structures of the synthesized hydrazone derivatives was achieved through the analysis of NMR, IR, and mass spectrometry data. All the newly prepared compounds were assessed for their in vitro α-glucosidase inhibition profiles and the results revealed a diverse range of inhibitory potentials, ranging from 9.3 ± 1.0 to 70.5 ± 1.8 μM, in contrast to the reference acarbose (with an IC50 value of 45.2 ± 1.3 μM). In an attempt to explain the observed inhibitions of synthesized naphthalene containing thiazole compounds, molecular docking was performed to determine the binding modes between the synthesized compounds from one side and the active residues of α-glucosidase from the other side. Compounds 4d, 4e, 4c, and 8 demonstrated the strongest binding affinities, with binding energies of -10.5, -9.8, -9.6, and -9.5 kcal/mol, respectively.

A Synbiotic Combining Chitin-Glucan and Lactobacillus acidophilus NCFM Induces a Colonic Molecular Signature Soothing Intestinal Pain and Inflammation in an Animal Model of IBS.

Chitin-glucan (CG) is a new generation of prebiotic. Lactobacillus acidophilus NCFM® (NCFM) is a probiotic with the ability to decrease abdominal pain. We evaluate the functional and molecular gastrointestinal responses to a synbiotic administration combining CG and NCFM in a rat model of long-lasting colon hypersensitivity. The intracolonic pressure was assessed during the 9-week experiment in animals receiving CG in association or not with NCFM and compared to that in Lacticaseibacillus paracasei Lpc-37®-treated animals and control rats receiving tap water. The effects of the synbiotic were evaluated using the Wallace score, the quantification of colon myeloperoxidase (MPO) and the master genes driving analgesia and inflammation. CG 1.5 alone and NCFM 109 colony forming units (CFU) alone similarly decreased the visceral pain sensitivity. Lpc-37 had no significant effect. The best profile of pain perception inhibition was obtained with the combination of CG 1.5 g and NCFM 109 CFU, confirming a synbiotic property. This synbiotic treatment significantly reduced macroscopic colonic lesions and MPO concentrations, and induced master genes involved in analgesia (CB1, CB2, MOR, PPARα), with a downregulation of inflammatory cytokines (IL-1β, TNFα) and an induction of IL-10 and PPARγ. In conclusion, CG 1.5 g + NCFM 109 CFU significantly decreased visceral pain perception and intestinal inflammation through the regulation of master genes.

Kinetic properties of gill (Na+, K+)-ATPase in the Pacific whiteleg shrimp Penaeus vannamei (Decapoda, Penaeidae)

The whiteleg marine shrimp Penaeus vannamei, originally from the Eastern Pacific Ocean, now inhabits tropical waters across Asia and Central and Southern America. This benthic species exhibits rapid growth, wide salinity and temperature tolerance, and disease resistance. These physiological traits have led to extensive research on its osmoregulatory mechanisms, including next-generation sequencing, transcriptomic analyses, and lipidomic responses. In crustaceans, osmotic and ionic homeostasis is primarily maintained by the membrane-bound metalloenzyme (Na+, K+)-ATPase. However, little is known about how various ligands modulate this enzyme in P. vannamei. Here, we examined the kinetic characteristics of the gill (Na+, K+)-ATPase to get biochemical insights into its modulation. A prominent immunoreactive band of ~120 kDa, corresponding to the (Na+, K+)-ATPase alpha-subunit, was identified. The enzyme exhibited two ATP hydrolyzing sites with K0.5 = 0.0003 ± 0.00002 and 0.05 ± 0.003 mmol L−1 and was stimulated by low sodium ion concentrations. Potassium and ammonium ions also stimulated enzyme activity with similar K0.5 values of 0.08 ± 0.004 and 0.06 ± 0.003 mmol L−1, respectively. Ouabain inhibition profile suggested a single enzyme isoform with a KI value of 2.10 ± 0.16 mmol L−1. Our findings showed significant kinetic differences in the (Na+, K+)-ATPase in Penaeus vannamei compared to marine and freshwater crustaceans. We expect our results to enhance understanding of the modulation of gill (Na+, K+)-ATPase in Penaeus vannamei and to provide a valuable tool for studying the shrimp's biochemical acclimation to varying salinity conditions.

Exploring Benzo[b][1,4]Thiazine Derivatives: Multitarget Inhibition, Structure–Activity Relationship, Molecular Docking, and ADMET Analysis

AbstractA series of benzothiazine derivatives (1–17) were synthesized via an intermolecular cyclocondensation reaction involving 2‐aminothiophenol (i) and substituted phenacyl bromide (ii). Structural elucidation of these synthetic derivatives utilized EI–MS, HR‐EIMS, 1H NMR, and 13C NMR spectroscopic techniques. The synthesized analogs were evaluated against key enzyme targets (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α‐glucosidase (α‐Glu)) and tested for cytotoxicity against various cancer cell lines. Six compounds were selected based on their inhibition profiles, exhibiting significant inhibitory potential against enzymes. In silico studies corroborated the observed inhibitory activities, aligning closely with experimental outcomes. Additionally, an ADME/T study provided insights into pharmacokinetic and safety profiles, identifying promising candidates for future drug development efforts.

Phosphorylation strongly affects the inhibition of human carbonic anhydrase I CO2 hydration activity

Human carbonic anhydrases (hCAs) have essential roles in respiration, acid-base balance, and fluid secretion, with implications in diseases such as glaucoma, epilepsy, obesity, and cancer. Of the fifteen known hCAs, human CA I (hCA I) is particularly abundant in erythrocytes, playing a critical role in CO2 transport. Despite extensive research on hCA I, the impact of post-translational modifications (PTMs), particularly phosphorylation, on its catalytic activity and inhibitor binding remains poorly understood. Although multiple phosphorylation sites have been identified in hCA I in vivo through high-throughput proteomics studies including at the highly conserved Ser51 residue, the functional consequences of these modifications are not well characterized. We investigated the effects of a phosphomimetic mutation at Ser51 on hCA I, examining its catalytic efficiency and susceptibility to inhibition by sulfonamides and anions. Using a recombinant expression system and a stopped-flow kinetic assay, we characterized the CO2 hydration activity and inhibition profiles of S51E hCA I compared to the wild type enzyme. Our results demonstrate that the S51E mutation increases the catalytic turnover rate (kcat) from 2.0 × 105 s−1 to 2.6 × 105 s−1 but significantly decreases substrate affinity, raising the Michaelis constant (KM) from 4.0 mM to 13.9 mM, reducing overall catalytic efficiency by over 50 %. Inhibition studies with a panel of 41 sulfonamides revealed that the S51E mutation dramatically alters inhibitor sensitivity, particularly for the most effective inhibitors. For example, 15 of the 16 most effective sulfonamide inhibitors for hCA I (with KIs <350 nM) were an average of over 35-fold less effective in inhibiting S51E hCA I than the wild type. The KI of the anticonvulsant zonisamide increased from 31 nM for the wild type hCA I to 4.0 μM. The inhibition profile with a panel of 37 small anions further indicated that the S51E mutant exhibited significantly reduced susceptibility to inhibition by 24 out of 37 tested anions, with some KI values increasing by up to 11,000-fold for inhibitors like hydrogen sulfide. This study underscores the significant impact that phosphorylation may have on hCA I function and inhibition. By characterizing the effects of phosphorylation on the CO2 hydration activity and inhibitor sensitivity of hCA I, these findings represent early steps in developing more selective proteoform-specific inhibitors, which could lead to more effective treatments for diseases involving carbonic anhydrases.