Natural Ligand Research Articles

8638 NEGR1 is a Mediator of Exercise-Induced Neuritogenesis in Subcutaneous White Adipose Tissue, Implications for Improved Systemic Metabolism

Abstract Disclosure: P. Nigro: None. N.P. Carbone: None. T. Caputo: None. M. Vamvini: None. M.M. Columbus: None. M.F. Hirshman: None. R. Middelbeek: None. L.J. Goodyear: None. Emerging research shows that exercise training results in robust and beneficial adaptions to subcutaneous white adipose tissue (scWAT), leading to improved systemic metabolism. The mechanisms for these benefits of exercise on are not fully understood, but we have recently demonstrated that training in mouse scWAT refines nerve connections in scWAT promoting neurite outgrowth (neuritogenesis). Our multi-omics and advanced microscopy data identifies Neuronal Growth Regulator 1 (NEGR1) as a potential player in this process. Here, we determine the role of NEGR1 as a mediator of scWAT neuritogenesis and regulator of systemic metabolism. Exercise training in mice for 11 days significantly increased NEGR1 protein expression by 35% (p<0.05). Next, we directly injected NEGR1 AAV into the inguinal scWAT of untrained male mice for three weeks resulting in a 43% increase in NEGR1 protein (p<0.05). Overexpression of NEGR1 led to an increase in the innervation markers tyrosine hydroxylase (+38%, p<0.01) and synaptophysin (+39%, p<0.01), and remarkably, a 16% decrease in adipocyte cell size (p<0.05), and a 15% reduction in fasting blood glucose levels (p<0.05). This suggests that NEGR1 plays a fundamental role in regulating scWAT morphology and function, adaptations that improve systemic glucose homeostasis. To determine mechanisms for exercise training-induced increases in NEGR1 expression, we used in silico and cell culture experiments and found that NEGR1 is under the regulation of the Peroxisome Proliferator-Activated Receptor gamma (PPARg) transcription factor. Given that numerous natural and synthetic PPARg ligands are lipids, we used targeted lipidomics to identify exercise-induced lipids capable of binding and inducing PPARg expression in inguinal scWAT. We identified 13-oxo-octadecadienoic acid (13-oxo-ODE), an oxylipin derived from linoleic acid, as a potent agonist and inducer of PPARg expression. Exercise training increased 13-oxo-ODE by 3-fold (p<0.05), and 13-hydroxyoctadecadienoic acid (13-HODE), the 13-oxo-ODE precursor was also increased by 3-fold (p<0.01). To validate that 13-oxo-ODE increases NEGR1, adipocytes were incubated with 2μM of 13-oxo-ODE for 48 hours. 13-oxo-ODE increased NEGR1 expression by 27-fold (p<0.0001) and NEGR1 protein by 2-fold (p<0.01). In summary, NEGR1 is a novel exercise-induced molecule that regulates neuronal refinement in scWAT. Our data also suggest that the synthesis of 13-oxo-ODE leads to PPARγ activation that in turn promotes NEGR1 expression in scWAT. NEGR1 has beneficial effects on scWAT that are associated with improved systemic metabolism, making NEGR1 a novel target for treatment of obesity and cardiometabolic diseases. Presentation: 6/1/2024

Functional characterization of CCHamides and deorphanization of their receptors in the yellow fever mosquito, Aedes aegypti

As a widely distributed anthropophilic mosquito species and vector of various arboviruses, Aedes aegypti poses a significant threat to human health on a global scale. Investigating mosquito neuropeptides allows us to better understand their physiology. The neuropeptides CCHamide1 (CCHa1) and CCHamide2 (CCHa2) along with their associated G protein-coupled receptors (CCHa1R and CCHa2R) were recently identified and studied across insects. However, expression profiles and physiological roles of CCHamides and their receptors in many other insects, including A. aegypti, remain unclear. This research aimed to quantify and localize the expression of CCHamides along with their receptors and gain insight on their physiological function in the yellow fever mosquito. RT-qPCR analysis revealed transcript abundance of CCHamides and receptors changes over development. Differential expression was also observed in tissues/organs of adult mosquitoes indicating CCHa1 and CCHa2 transcripts are enriched in the midgut, while receptors are expressed across various tissues. CCHamide immunoreactivity was observed in neurons in the brain and ventral nerve cord along with enteroendocrine cells in the posterior midgut adjacent to the midgut-hindgut junction, corroborating their transcript expression profiles. Using different mass spectrometrical approaches, presence of CCHamides were confirmed in the brain of both sexes, including the pars intercerebralis of female mosquitoes, as well as in the gut of adult mosquitoes. For chemical identification of predicted CCHamides, we analyzed brain and gut extracts by ESI-Q Exactive Orbitrap MS and resulting fragmentations confirmed CCHa1 and CCHa2 in brain and midgut samples of both male and female mosquitoes. A heterologous functional assay was used to confirm the specificity and sensitivity of the two CCHamide receptors by assessing their activation in response to diverse mosquito peptides, which confirmed CCHa1 and CCHa2 as natural ligands. Finally, using a capillary feeder (CAFE) bioassay, our results suggest that CCHa2 modulates feeding behaviour in female mosquitoes.

B-343 Development of CD40 and HER2 Bispecific Antibodies for Enhanced Efficacy against HER2-Positive Tumors

Abstract Background CD40, a member of the tumor necrosis factor superfamily, is primarily expressed on antigen-presenting cells. This receptor plays a crucial role in facilitating the interaction between dendritic cells, activating antigen-specific T cells. Agonist antibodies targeting the CD40 receptor have the potential to mimic the natural CD40 ligand, fostering dendritic cell maturation and stimulating immunity against “cold” tumors. Although this approach has shown promise in preclinical studies, translating it to clinical success has been challenging. Only modest anti-tumor activity was observed in cancer patients, and none of the developed CD40 monoclonal antibodies (mAbs) progressed beyond early clinical trials, highlighting a need for improved anti-tumor efficacy. Methods In this study, we successfully developed an anti-human CD40 agonist rabbit monoclonal antibody using Yurogen's proprietary SMAB(TM) platform. The most potent CD40 mAb clone was combined with trastuzumab (an anti-HER2 mAb) to create a therapeutic bispecific antibody. Our goal is to leverage immune cell activation through HER2-targeted cancer therapy to achieve enhanced anti-tumor activity. Dozens of diverse CD40xHER2 bispecific antibody designs (both symmetry and asymmetry) were explored, with careful evaluation of expression yield, purity, and binding profiles for each target respectively. Results A few bispecific antibody designs demonstrated robust binding to both HER2 and CD40 antigen proteins, as well as receptor-expressing cell lines. Notably, two developed bispecific antibodies exhibited potent killing of HER2-positive cancer cells with the assistance of innate immune cells. Conclusions In sum, this study provides novel insights into designing bispecific antibodies against HER2 and CD40, offering a promising avenue for advancing cancer immunotherapy.

A platform for the early selection of non-competitive antibody-fragments from yeast surface display libraries.

In this work, we report the development of a platform for the early selection of non-competitive antibody-fragments against cell surface receptors that do not compete for binding of their natural ligand. For the isolation of such subtype of blocking antibody-fragments, we applied special fluorescence-activated cell sorting strategies for antibody fragments isolation from yeast surface display libraries. Given that most of the monoclonal antibodies approved on the market are blocking ligand-receptor interactions often leading to resistance and/or side effects, targeting allosteric sites represents a promising mechanism of action to open new avenues for treatment. To directly identify these antibody-fragments during library screening, we employed immune libraries targeting the epidermal growth factor receptor as proof of concept. Incorporating a labeled orthosteric ligand during library sorting enables the early selection of non-competitive binders and introduces an additional criterion to refine the selection of candidates exhibiting noteworthy properties. Furthermore, after sequencing, more candidates were identified compared to classical sorting based solely on target binding. Hence, this platform can significantly improve the drug discovery process by the early selection of more candidates with desired properties.

Structural basis of selective beta-carotene binding by a soluble protein

β-carotene (BCR) is the most abundant carotenoid, a colorant, antioxidant, and provitamin A. The extreme hydrophobicity of this hydrocarbon requires special mechanisms for distribution in aqueous media, including water-soluble carotenoproteins. However, all known carotenoproteins prefer oxygenated carotenoids and bind BCR inefficiently. Here, we present the crystal structure of the BCR-binding protein (BBP) from gregarious male locusts, which is responsible for their vivid yellow body coloration, in complex with its natural ligand, BCR. BBP forms an antiparallel tubular homodimer with α/β-wrap folded monomers, each forming a hydrophobic 47Å long, coaxial tunnel that opens outward and is occupied by one s-cisC6-C7, all-trans BCR molecule. In the BCR absence, BBP accepts a range of xanthophylls, with reduced efficiency depending on the position and number of oxygen atoms, but rejects lycopene. The structure captures a pigment complex with a Takeout 1 protein and inspires potential applications of BBP as a BCR solubilizer.

Rationale design and synthesis of new roflumilast analogues as preferential selective and potent PDE-4B inhibitors

In this study, we designed and synthesized novel analogues of roflumilast that exhibit selective inhibition of PDE-4B. To accomplish this target; synthesis of novel series (4a-u, 5a-i, and 6) was done, aiming at obtaining new PDE-4B inhibitors hits based on the proposed pharmacophore, 1-(cyclopropylmethoxy)-2-(difluoromethoxy) benzene moiety. Enzyme assay was used to measure the IC50 values for the PDE-4B inhibition of all the synthesized compounds along with roflumilast as a reference drug. The results demonstrated that most of the examined candidates exhibited considerable inhibitory activity against the PDE-4B enzyme. The four compounds (4i, 4k, 4p, and 4q) exhibited the highest potency (IC50 = 7.25, 7.15, 5.50, 7.19 nM, respectively) with no significant inhibition difference from roflumilast (no statistical difference at p < 0.05). Interestingly, compound 4p with 3-OH and 4-OCH3 substituents was found to be the most potent against PDE-4B enzyme (IC50 = 5.50 nM), compared to that of roflumilast (IC50 = 2.36 nM). Moreover, the most potent derivatives 4i, 4k, 4p, and 4q were further tested for PDE-4D inhibitory activity to investigate their PDE-4D/PDE-4B selectivity ratio. Compound 4k showed the highest selectivity towards PDE-4B isozyme more than the reference drug roflumilast (PDE-4D/4B IC50 ratio for compound 4k and roflumilast = 3.22 and 3.02, respectively). Additionally, compound 4p was chosen to test its selectivity for PDE-4B over PDE-8A, PDE-11A, and PDE-1B compared to thereference drug roflumilast. Compound 4p showed approximately 6-fold selectivity for PDE-4B over PDE-8A, about 5-fold selectivity for PDE-4B over PDE-11A, and 11-fold selectivity of PDE-4B over PDE-1B. Compound 4p showed a higher selectivity towards PDE-4B than PDE-1B, more than the reference compound roflumilast. Furthermore, the most potent compounds (4i, 4k, 4p, 4q) were subjected to further investigation, and their effects on the cAMP level and percentage of inhibition of tumor necrosis factor-alpha (TNF-α) were studied and compared with reference drug roflumilast. Compound 4q showed the highest increase in the level of intracellular cAMP (6.55 ± 0.37 pmol/mL) and compound 4i showed the highest % of TNF-α inhibition (77.22 %). On the other side, a molecular docking study against PDE-4B clarified that all the examined candidates achieved nearly similar binding modes with similar orientations to that of the native roflumilast ligand and showed higher docking scores than roflumilast.

Phytobioinformatics screening of ayurvedic plants for potential α-glucosidase inhibitors in diabetes management

The enzyme α-glucosidase in the small intestine regulates blood glucose levels and stimulates the hydrolysis of oligosaccharides and polysaccharides, increasing glucose levels in the body. Inhibiting this enzyme slows glucose digestion and absorption and as a result post-prandial blood glucose levels remain low, causing decreased insulin demand. Here, we investigated the ayurvedic antidiabetic plants and virtually screened an in-house library of 478 phytochemicals of these plants against the human α-glucosidase. We identified 11 secondary metabolites, including palmitic acid α-monoglyceride, (+)-(2 R)-6-propionyloxyethyl-4′,5,7-trihydroxyisoflavanone, Abruquinone E, and Aurantiamide Acetate, among others, showed stronger interactions with the receptor than the native ligand N-acetyl cysteine. Surprisingly, except one, all of these metabolites were from Abrus precatorius L. [Fabaceae] affirming its ethnopharmacological use against diabetes. The stability of the interactions between the ligands and receptor protein was evaluated through Molecular Dynamic (MD) simulation trajectories including root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), H bonds, β-factor analysis, and binding energy calculation through MM/GBSA method. The efficacy of top metabolites in inhibiting α-glucosidase is depicted in pharmacophore analysis. A comprehensive pharmacokinetics analysis confirmed the druggability, safety, and efficiency of top drug candidates. Additionally, we predicted the interactions of these top metabolites within the biological system. The medicinal properties described in this study will help develop active drug candidates for therapeutic purposes. Further experiments are recommended to prove the effectiveness of these metabolites in inhibiting the α-glucosidase enzyme for exploring their potential in the treatment of diabetes.

Unveiling neuroprotective mechanisms of diosgenin and pterostilbene in diabetes-associated Alzheimer’s disease through multi-target molecular docking approach

Introduction: Diabetes mellitus (DM) and Alzheimer’s disease (AD) are two highly linked disorders due to their association with the aging population. Several studies have reported the beneficial effects of diosgenin and pterostilbene in treating neurodegenerative diseases. This study aimed to investigate the neuroprotective mechanisms of diosgenin and pterostilbene through molecular docking and dynamics studies and assess their pharmacokinetic parameters. Methods: To understand the link between diabetes and AD, molecular docking of the natural ligands diosgenin and pterostilbene against the specific targets of AD, including β-secretase, glycogen synthetase kinase beta (GSK-3β), gamma-secretase, tumor necrosis factor-alpha (TNF-ɑ), and interleukin-6 (IL-6) was done to find out their binding affinities to explain the molecular mechanisms involved in their neuroprotection. Further molecular dynamics studies and binding energy calculations for the ligands with GSK-3β and β-secretase were carried out to confirm their docking activities. Additionally, pharmacokinetic analysis of these two phytoconstituents was performed by the SWISSADME server. Results: Molecular docking and dynamics studies revealed the good docking activity of diosgenin and pterostilbene with the selected targets. Further, the two phytoconstituents revealed suitable pharmacokinetic parameters along with blood-brain barrier permeability, confirming their druggable nature. Conclusion: This research identified multiple neuroprotective pathways of diosgenin and pterostilbene that might be significant for treating diabetes-associated Alzheimer’s disease.

Assembly of 2′,3′-Cyclic guanosine Monophosphate-Adenosine monophosphate and their spontaneous intracellular disassembly for enhanced antitumor immunity via natural STING pathway activation

The stimulator of interferon genes (STING) pathway plays an important role in remodeling the immunosuppressive tumor microenvironment (TME). Cyclic dinucleotides (CDNs), the natural ligands of STING, activate innate immunity to enhance tumor immunogenicity. However, the anionic properties of CDNs result in poor membrane permeability, while their rapid metabolism by enzymes hinders the efficient targeting and activation of STING in vitro and in vivo. To improve the bioavailability and antitumor immunity of CDNs, we designed a hydrogen-bonding-based supramolecular approach for 2′,3′-cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP). Leveraging the synthetic molecules cytosine-uracil-hexane (CUH), we formulated supramolecular micelles of cGAMP via specific hydrogen bonds in aqueous solutions. Micelles comprising the CUH-cGAMP complex enhanced the biological efficacy of cGAMP by increasing its stability, thereby prolonging the activation of STING-mediated innate and adaptive immunity. We demonstrated that CUH-cGAMP stands as a promising candidate for immunotherapy, exhibiting significant inhibition of tumor growth in a CT26 syngeneic mouse model.

Design and In-silico Study of ¹³¹Iodium Radiolabeled of Thiourea derivatives for Breast Cancer Treatment

Breast cancer is a serious challenge in both developed and developing countries, with current therapies still limited and potentially producing adverse side effects. To optimize breast cancer drug development, this study adopted an in-silico design approach. The aim is to develop radiopharmaceutical drugs with minimal side effects. Methods involved molecular docking analysis, molecular dynamics, drug scan, and pharmacokinetic profile prediction of the original ligan as well as the radioligand. Results showed that the radioligand had better binding energy and inhibition constant than tamoxifen as a comparator drug which is -11.31 kcal/mol and 0.00511 µM. Molecular dynamics analysis revealed that the radioligand compound exhibits comparable RMSD, RMSF, and stability metrics to the native ligand at the GRPR receptor with average RMSD and RMSF of 5.263 Å and 2.285 Å, respectively. By considering the results of these various methods, the radioligand compound shows potential as an effective radiopharmaceutical drug in breast cancer therapy.

&amp;lt;i&amp;gt;In-Silico&amp;lt;/i&amp;gt; Screening of Prenylated Quercetin from &amp;lt;i&amp;gt;Globimetula oreophila&amp;lt;/i&amp;gt; Against &amp;lt;i&amp;gt;Plasmodium falciparum&amp;lt;/i&amp;gt; Enzymes: Hope for New Antimalarial Drugs

Malaria continues to be one of the most devastating global health problems due to the high morbidity and mortality it causes in endemic regions. The search for new antimalarial targets is vital because of the increasing prevalence of drug resistance in malaria parasites. Malarial proteases constitute promising therapeutic targets as they play important roles in the parasite life cycle. The inhibition of these enzymes has pharmacological and therapeutic significance since they are involved in numerous processes, including the development, invasion, egress, and breakdown of host hemoglobin to release amino acids for parasite sustenance. In this study, &amp;lt;i&amp;gt;in silico&amp;lt;/i&amp;gt; techniques were used to shed light on the mechanisms underlying the inhibitory effects of prenylated quercetin isolated from &amp;lt;i&amp;gt;Globimetula oreophila&amp;lt;/i&amp;gt; on plasmepsin I and II, falciparum 2 and 3, &amp;lt;i&amp;gt;Plasmodium falciparum&amp;lt;/i&amp;gt; calcium-dependent protein kinase 2, dihydrofolate reductase-thymidylate synthase, and serine repeat antigen 5. The test compound significantly interacts with key enzyme binding pockets through hydrogen bonds, van der Waals, and hydrophobic interactions, influencing protease specificity control. Crucial ligand features like carbonyl and hydroxyl groups were identified as essential for receptor interactions. Comparative analysis revealed the test compound&amp;apos;s strong binding affinities with energies ranging from -6.4 Kcal/mol to -9.4 Kcal/mol, indicating competitive potential against various enzymes, particularly excelling against &amp;lt;i&amp;gt;Pf&amp;lt;/i&amp;gt;DHFR-TS, plasmepsin-I, and SERA5 compared to native ligands. This suggests the compound&amp;apos;s ability to competitively inhibit enzyme activity by targeting co-factor binding sites, especially with specific proteases, holding promise for therapeutic applications as potent inhibitors for the prevention and treatment of malaria.

Influence of hyaluronic acid and chitosan molecular weight on the adhesion of circulating tumor cell on multilayer films

CD44 is a cell receptor glycoprotein overexpressed in circulating tumor cells (CTCs), with levels linked to an increase in metastatic capacity of several tumors. Hyaluronic acid (HA), the natural ligand of CD44, has primarily been investigated for tumor cell interaction in self-assembled polyelectrolyte multilayer films, with little attention given to the complementary polycation. In this study, we screened sixteen different polyelectrolyte multilayer assemblies of HA and chitosan (CHI) to identify key assembly parameters and surface properties that control and govern CTCs adhesion. Statistics analysis revealed a major role of CHI molecular weight in the adhesion, followed by its combinatorial response either with HA ionization degree or ionic strength. PM-IRRAS analysis demonstrated a correlation between the orientation of HA carboxyl groups on the film surface and CTCs adhesion, directly impacted by CHI molecular weight. Overall, although CTCs binding onto the surface of multilayer films is primarily driven by HA-CD44 interaction, both chitosan properties and film assembly conditions modulate this interaction. These findings illustrate an alternative to modifying the performance of biomaterials with minimal changes in the composition of multilayer films.

Designed endocytosis-inducing proteins degrade targets and amplify signals.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificitytargeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates.

Investigating In Silico and In Vitro Therapeutic Potential of Diosmetin as the Anti-Parkinson Agent.

This study aimed to investigate how diosmetin interacts with seven target receptors associated with oxidative stress (OS) and validate its antioxidant properties for the potential management of Parkinson's disease (PD). In PD, the degeneration of dopaminergic cells is strongly influenced by OS. This stressor is intricately connected to various mechanisms involved in neurodegeneration, such as mitochondrial dysfunction, neuroinflammation, and excitotoxicity induced by nitric oxide. The aim of this research was to establish a molecular connection between diosmetin and OS-associated target receptors was the goal, and it investigated how this interaction can lessen PD. Seven molecular targets - Adenosine A2A (AA2A), Peroxisome Proliferator- Activated Receptor Gamma (PPARγ), Protein Kinase AKT1, Nucleolar Receptor NURR1, Liver - X Receptor Beta (LXRβ), Monoamine Oxidase - B (MAO-B) and Tropomyosin receptor kinase B (TrkB) were obtained from RCSB. Molecular docking software was employed to determine molecular interactions, while antioxidant activity was assessed through in-vitro assays against various free radicals. Diosmetin exhibited interactions with all seven target receptors at their binding sites. Notably, it showed superior interaction with AA2A and NURR1 compared to native ligands, with binding energies of -7.55, and -6.34 kcal/mol, respectively. Additionally, significant interactions were observed with PPARγ, AKT1, LXRβ, MAO-B, and TrkB with binding energies of -8.34, -5.42, -7.66, -8.82, -8.45 kcal/mol, respectively. Diosmetin also demonstrated antioxidant activity against various free radicals, particularly against hypochlorous acid (HOCl) and nitric oxide (NO) free radicals. Diosmetin possibly acts on several target receptors linked to the pathophysiology of PD, demonstrating promise as an OS inhibitor and scavenger.

Kajian Farmakoinformatika Senyawa Caulerchlorin dan Racemosin sebagai kandidat Antimalaria

Caulerchlorin and racemosin were bioactive compounds containing in Caulerpa racemosa macroalgae. Previous study, those compounds promoted therapeutical target on metabolic syndrome. However, the therapeutical activity on Malaria not yet defined. Therefore, this study investigated potential activity of caulerchlorin and racemosin from Caulerpa racemosa as antimalarial activity through pharmacoinformatic study. Caulerchlorin and racemosin structures were retrieved from PubChem NCBI and the Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) structure was taken out from Protein database with ID 5GJG. Orotic acid was used as native ligand or control. Ligands were predicted their antiplasmodial activity and toxicity. Ligands and protein were interacted by redocking using Molegro Virtual Docker version 6.0 and analyzed by using PyMol 2.3 and Discovery Studio version 21.1.1. Two bioactive compounds also showed antiplasmodial activity and were not mutagenicity and hepatotoxicity affections. Molecular docking performed that caulerchlorin and racemosin exhibited same active residues as well as orotic acid as native substrate. Binding energy revealed two compounds, caulerchlorin and racemosin showed lower binding energy than orotic acid. In summary caulerchlorin and racemosin were potentially inhibited PfDHODH activity by binding substrate sites of PfDHODH protein.

Flavokawain B is an effective natural peroxisome proliferator-activated receptor γ-selective agonist with a strong glucose-lowering effect

Rosiglitazone, a full PPARγ agonist and a classical insulin sensitizer, was once used as a powerful weapon in the treatment of T2DM. However, its applications have been restricted recently because of its multiple side effects. Here, a natural compound, flavokawain B (FKB), which was screened in our previous experiments, was investigated for its potential as a preferable insulin sensitizer because it has no or few side effects. Using the surface plasmon resonance (SPR) technique, we confirmed that FKB is a natural ligand for PPARγ with high binding affinity. In in vitro experiments, FKB significantly increased 2-NBDG uptake in HepG2 and 3T3-L1 cells, which partially stimulated PPARγ transcriptional activity. Compared with rosiglitazone, FKB had little effect on the adipose differentiation of 3T3-L1 cells, and all of these features suggest that FKB is a selective modulator of PPARγ (SPPARγM). Moreover, FKB increased the mRNA expression levels of most genes related to insulin sensitivity and glucose metabolism but had no obvious effect on those related to adipose differentiation. In vivo experiments confirmed that FKB effectively decreased abnormal fasting blood glucose and postprandial blood glucose levels and reduced glycated hemoglobin levels, similar to rosiglitazone, in HFD-fed/STZ-treated and db/db mice, two T2DM animal models, but did not cause side effects, such as weight gain or liver or kidney damage. Further investigation revealed that FKB could inhibit PPARγ-Ser273 phosphorylation, which is the key mechanism involved in improving insulin resistance. Together, FKB is a well-performing SPPARγM that exerts a powerful glucose-lowering effect without causing the same side effects as rosiglitazone, and it may have great potential for development.

ARGX-119 is an agonist antibody for human MuSK that reverses disease relapse in a mouse model of congenital myasthenic syndrome.

Muscle-specific kinase (MuSK) is essential for the formation, function, and preservation of neuromuscular synapses. Activation of MuSK by a MuSK agonist antibody may stabilize or improve the function of the neuromuscular junction (NMJ) in patients with disorders of the NMJ, such as congenital myasthenia (CM). Here, we generated and characterized ARGX-119, a first-in-class humanized agonist monoclonal antibody specific for MuSK, that is being developed for treatment of patients with neuromuscular diseases. We performed in vitro ligand-binding assays to show that ARGX-119 binds with high affinity to the Frizzled-like domain of human, nonhuman primate, rat, and mouse MuSK, without off-target binding, making it suitable for clinical development. Within the Fc region, ARGX-119 harbors L234A and L235A mutations to diminish potential immune-activating effector functions. Its mode of action is to activate MuSK, without interfering with its natural ligand neural Agrin, and cluster acetylcholine receptors in a dose-dependent manner, thereby stabilizing neuromuscular function. In a mouse model of DOK7 CM, ARGX-119 prevented early postnatal lethality and reversed disease relapse in adult Dok7 CM mice by restoring neuromuscular function and reducing muscle weakness and fatigability in a dose-dependent manner. Pharmacokinetic studies in nonhuman primates, rats, and mice revealed a nonlinear PK behavior of ARGX-119, indicative of target-mediated drug disposition and in vivo target engagement. On the basis of this proof-of-concept study, ARGX-119 has the potential to alleviate neuromuscular diseases hallmarked by impaired neuromuscular synaptic function, warranting further clinical development.

Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Structure-Activity Relationship of Inositol Thiophosphate Analogs as Allosteric Activators of Clostridioides difficile Toxin B.

Clostridioides difficile is a bacterium that causes life-threatening intestinal infections. Infection symptoms are mediated by a toxin secreted by the bacterium. Toxin pathogenesis is modulated by the intracellular molecule, inositol-hexakisphosphate (IP6). IP6 binds to a cysteine protease domain (CPD) on the toxin, inducing autoproteolysis, which liberates a virulence factor in the cell cytosol. We developed second-generation IP6 analogs designed to induce autoproteolysis in the gut lumen, prior to toxin uptake, circumventing pathogenesis. We synthesized a panel of thiophosphate-/sulfate-containing IP6 analogs and characterized their toxin binding affinity, autoproteolysis induction, and cation interactions. Our top candidate was soluble in extracellular cation concentrations, unlike IP6. The IP6 analogs were more negatively charged than IP6, which improved affinity and stabilization of the CPD, enhancing toxin autoproteolysis. Our data illustrate the optimization of IP6 with thiophosphate biomimetic which are more capable of inducing toxin autoproteolysis than the native ligand, warranting further studies in vivo.

Analysis of cell signaling profiles induced by DNA aptamer-based FGFR1 agonist.

DNA aptamers have attracted attention as an alternative modality for biomolecules due to their excellent target binding specificity and thermal stability, and they are also expected to be applied as artificial agonists for receptor proteins. DNA aptamer agonist TD0 targeting the receptor of fibroblast growth factor (FGFR), which plays an important role in the fields of wound healing and regenerative medicine, has been reported to induce cellular responses as well as its native ligands. However, it was also noted that there were some different responses upon long-term stimulation, suggesting that the intracellular signals induced by DNA aptamer agonist TD0 are different from those of natural ligands. In this paper, we comprehensively analyzed the intracellular signals induced by DNA aptamer agonist TD0 targeting FGFR1, and compared them with those by natural protein ligand FGF2. It was found that the intracellular signals were highly similar for short-term stimulation. On the other hand, the receptor and the downstream cellular signals showed different activation behaviors for long-time stimulation. Evaluating the stability and sustained activity of DNA aptamer agonist TD0 and FGF2 in the medium suggested that ligand stability may be important in properly regulating cellular responses.