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BACKGROUND: The widespread evolution of pesticide resistance poses a significant challenge to current agriculture, necessitating the discovery of molecules with new modes of action. Despite extensive efforts, no major molecules with new modes of action have been commercialized for decades. Most pesticides function by binding to specific pockets on target enzymes, enabling a single target site mutation to confer resistance. An alternative approach is the disruption of protein-protein interactions (PPI), which require complementary mutations on both interacting partners for resistance to occur. Thus, our aim is the discovery and design of small-molecule inhibitors that target the interface of the PPI complex of O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT), key obligatory interacting plant enzymes involved in the biosynthesis of the amino acid cysteine. RESULTS: By employing in silico filtering techniques on a virtual library of 30 million small molecules, we identified initial hits capable of binding OASS and interfering with its interaction with a peptide derived from SAT with a half-maximal inhibitory concentration (IC50) of 34 µm. Subsequently, we conducted molecular chemical optimizations, generating an early lead molecule (PJ4) with an IC50 value of 4 µm. PJ4 successfully inhibited the germination of Arabidopsis thaliana seedlings and inhibited clover growth in a pre-emergence application at an effective concentration of 4.6 kg ha-1. CONCLUSION: These new compounds described herein can serve as promising leads for further optimization as herbicides with a new mode-of-action. This technology can be used for discovering new modes of action chemicals inhibiting all pest groups. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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The synthesis of essential amino acids in plants is pivotal for their viability and growth, and these cellular pathways are therefore targeted for the discovery of new molecules for weed control. Herein, we describe the discovery and design of small molecule inhibitors of cystathionine gamma-synthase, a key enzyme in the biosynthesis of methionine. Based on in silico screening and filtering of a large molecular database followed by the in vitro selection of molecules, we identified small molecules capable of binding the target enzyme. Molecular modelling of the interaction and direct biophysical binding enabled us to explore a focussed chemical expansion set of molecules characterized by an active phenyl-benzamide chemical group. These molecules are bio-active and efficiently inhibit the viability of BY-2 tobacco cells and seedlings growth of Arabidopsis thaliana on agar plates.
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Arabidopsis , Liasas de Carbono-Oxígeno , Metionina , NicotianaRESUMEN
Immune-checkpoint receptors are a set of signal transduction proteins that can stimulate or inhibit specific anti-tumor responses. It is well established that cancer cells interact with different immune checkpoints to shut down T-cell response, thereby enabling cancer proliferation. Given the importance of immune checkpoint receptors, a structure-function analysis of these systems is imperative. However, recombinant expression and purification of these membrane originated proteins is still a challenge. Therefore, many attempts are being made to improve their expression and solubility while preserving their biological relevance. For this purpose, we designed an E. coli-based optimization system that enables the acquisition of mutations that increases protein solubility and affinity towards its native ligand, while maintaining biological activity. Here we focused on the well-characterized extracellular domain of the 'programmed cell death protein 1' (PD1), an immune checkpoint receptor known to inhibit T-cell proliferation by interacting with its ligands PD-L1 and PD-L2. The simple ELISA-based screening system shown here enabled the identification of high-affinity, highly soluble, functional variants derived from the extracellular domain of human PD1. The system was based on the expression of a GST-tagged variants library in E. coli, which enabled the selection of improved PD1 variants after a single optimization round. Within only two screening rounds, the most active variant showed a 5-fold higher affinity and 2.4-fold enhanced cellular activity as compared to the wild type protein. This scheme can be translated toward other types of challenging receptors toward development of research tools or alternative therapeutics.
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Antígeno B7-H1/metabolismo , Escherichia coli/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Biblioteca de Genes , Humanos , Receptor de Muerte Celular Programada 1/química , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , SolubilidadRESUMEN
Calcineurin is a phosphatase that targets the transcription factor, nuclear factor of activated T-cells (NFAT) dephosphorylates multiple sites along NFAT's regulatory domain. The calcineurin-NFAT complex interaction is mediated through two conserved binding motifs known as the PxIxIT and LxVP, which are located at the N- and C- terminus to the phosphorylation sites. The vast range of cellular processes regulated by the calcineurin-NFAT interaction has aroused great interest in the investigation of the structural aspects that govern their complex formation and in the discovery of protein-protein interaction inhibitors; the latter interfere with calcineurin-NFAT complex formation while keeping calcineurin's catalytic site free. To assist additional biophysical study of the calcineurin-NFAT structure-function relation and to screen for new inhibitors, we present a robust and cost-effective Enzyme Linked Immuno Sorbent Assay (ELISA) that is based on the interaction of calcineurin with the NFAT homology region. The latter includes the two calcineurin's binding sites, in addition to the phosphorylation sites. The ELISA experiment shown here can thus be applied towards the study of important structural aspects of the complex and for the discovery of new inhibitors. This will allow for a better understanding of T-cell activation switch.
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Calcineurina/química , Factores de Transcripción NFATC/química , Calcineurina/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , HumanosRESUMEN
Protein kinases are fundamental within almost all cellular signal transduction networks. Among these, Bruton's tyrosine kinase (Btk), which belongs to the Tec family of proteins, plays an imperative part in B-cell signaling. Owing to its role, Btk has been established as an important therapeutic target for a vast range of disorders related to B-cell development and function, such as the X-linked agammaglobulinemia, various B-cell malignancies, inflammation, and autoimmune diseases. Herein, using computer-based screening of a library of 20 million small molecules, we identified a small molecule capable of directly binding the Btk kinase domain. On the basis of this hit compound, we conducted a focused structure-similarity search to explore the effect of different chemical modifications on binding toward Btk. This search identified the molecule N2,N6-bis(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-9H-purine-2,6-diamine as a potent inhibitor of Btk. The latter small molecule binds Btk with a dissociation constant of 250 nM and inhibits Btk activity both in vitro and in-cell.
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Prostatic adenocarcinoma is the second leading cause of death from cancer in Western men. The common prostate cancer treatments are effective in the early stages; however, advanced prostate cancer is resilient to most of these treatments. Altered androgen receptor (AR) activity caused by point mutations or signaling mechanisms that regulate AR function has been proposed as a key mechanism in the transition to the androgen-independent stage. Our previous results demonstrated that hexane extract prepared from Coprinus comatus (C. comatus) strain 734 was able to interfere with AR activity. The current study was made to further evaluate the antiandrogenic activity of the C. comatus mushroom strain 734. Activity-guided chromatography was conducted and 2 active fractions, F-32-and F-33, were found to contain substances that were able to inhibit AR-mediated reporter activity and reduce the levels of AR and prostate-specific antigen (PSA) transcripts in LNCaP cells. Fraction F-32 also inhibited the proliferation and clonigenicity of LNCaP cells. Furthermore, F-32 was able to inhibit the binding of AR to the PSA enhancer region and to inhibit Akt-mediated AR phosphorylation at Ser 213. This study illustrated the potential of substances from the C. comatus mushroom to serve as natural antiandrogenic modulators for the treatment of prostatic disorders.
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Coprinus/química , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Antagonistas de Andrógenos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Fosforilación , Antígeno Prostático Específico/efectos de los fármacos , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Prostate cancer is the most common cancer diagnosed in men. Chemotherapy, androgen ablation, and androgen antagonist treatments have proven to have significant effects in the early stages of prostate cancer, whereas advanced prostate cancer is resilient to such treatments. The androgen receptor (AR), a ligand-dependent transcription factor, is the major drug target of prostate cancer therapy. Transition to the androgen-independent stage involves the activation of signaling pathways, AR gene mutations, and other mechanisms. Higher basidiomycetes mushrooms have been used since ancient times in folk medicine to treat a diversity of diseases, including cancer. The present study evaluates the antiandrogenic activity of different Coprinus comatus strains in their ability to interfere with AR function. The authors found that the most active extract was C comatus strain 734 extracted with hexane (CC734-H). This extract was able to (1) inhibit AR-mediated reporter activity, (2) inhibit the proliferation and viability of the LNCaP cell line, and (3) inhibit the colony formation of the LNCaP cell line, in comparison to the DU-145, PC-3, and MDA-Kb2 cells. In addition, CC734-H was able to reduce AR levels and prostate-specific antigen gene expression in the LNCaP-treated cell line. This study illustrates the potential of the C comatus mushroom as a natural antiandrogenic modulator that could serve in the treatment of prostatic diseases.
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Antagonistas de Andrógenos/farmacología , Coprinus/química , Extractos Vegetales/farmacología , Receptores Androgénicos/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Antagonistas de Andrógenos/aislamiento & purificación , Andrógenos/farmacología , Compuestos de Anilina/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dexametasona/farmacología , Dihidrotestosterona/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , Flutamida/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Genes Reporteros/genética , Humanos , Concentración 50 Inhibidora , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Mifepristona/farmacología , Nitrilos/farmacología , Extractos Vegetales/aislamiento & purificación , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Quinolinas/farmacología , Receptores Androgénicos/genética , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Ensayo de Tumor de Célula Madre , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Prostate cancer (PCa) is the most common male malignancy in many Western countries. Primary PCa is hormone dependent and is manageable by hormonal therapy. However, it rapidly develops to hormone-refractory tumors due to the accumulation of mutations in the androgen receptor and/or the acquisition of alternative cellular pathways that support proliferation and inhibit apoptosis of prostate cancer. To date, no effective therapy is available for clinically hormone-insensitive or hormone-refractory stages of prostate cancer.