Active Site Pocket Research Articles

Farnesol dehydrogenase from Helicoverpa armigera (Hübner) as a promising target for pest management: molecular docking, in vitro and insect bioassay studies using geranylgeraniol as potential inhibitor.

Juvenile hormone (JH) plays pivotal roles in several critical developmental processes in insects, including metamorphosis and reproduction. JH-biosynthetic pathway enzymes are considered highly promising targets for discovering novel insecticides. The oxidation of farnesol to farnesal, catalysed by farnesol dehydrogenase (FDL), represents a rate-limiting step in JH biosynthesis. Here, we report farnesol dehydrogenase (HaFDL) from H. armigera as a promising insecticidal target. The inhibitory potential of natural substrate analogue geranylgeraniol (GGol) was tested in vitro, wherein it showed a high binding affinity (kd 595µM) for HaFDL in isothermal titration calorimetry (ITC) and subsequently exhibited dose-dependent enzyme inhibition in GC-MS coupled qualitative enzyme inhibition assay. Moreover, the experimentally determined inhibitory activity of GGol was augmented by the in silico molecular docking simulation which showed that GGol formed a stable complex with HaFDL, occupied the active site pocket and interacted with key active site residues (Ser147 and Tyr162) as well as other residues that are crucial in determining the active site architecture. Further, the diet-incorporated oral feeding of GGol caused detrimental effects on larval growth and development, exhibiting a significantly reduced rate of larval weight gain (P < 0.01), aberrant pupal and adult morphogenesis, and a cumulative mortality of ~ 63%. To the best of our knowledge, the study presents the first report on evaluating GGol as a potential inhibitor for HaFDL. Overall, the findings revealed the suitability of HaFDL as a potential insecticidal target for the management H. armigera.

The T1150A cancer mutant of the protein lysine dimethyltransferase NSD2 can introduce H3K36 trimethylation

Protein lysine methyltransferases (PKMTs) play essential roles in gene expression regulation and cancer development. Somatic mutations in PKMTs are frequently observed in cancer cells. In biochemical experiments, we show here that the NSD1 mutations Y1971C, R2017Q and R2017L observed mostly in solid cancers are catalytically inactive suggesting that NSD1 acts as tumor suppressor gene in these tumors. In contrast, the frequently observed T1150A in NSD2 and its T2029A counterpart in NSD1, both observed in leukemia, are hyperactive and introduce up to thee methyl groups in H3K36 in biochemical and cellular assays, while wildtype NSD2 and NSD1 only introduce up to two methyl groups. In molecular dynamics simulations, we determine key mechanistic and structural features controlling the product specificity of this class of enzymes. Simulations with NSD2 revealed that H3K36me3 formation is possible due to an enlarged active site pocket of T1150A and loss of direct contacts of T1150 to critical residues which regulate the product specificity of NSD2. Bioinformatic analyses of published data suggested that the generation of H3K36me3 by NSD2 T1150A could alter gene regulation by antagonizing H3K27me3 finally leading to the upregulation of oncogenes.

Allosteric Inhibitors of Zika Virus NS2B-NS3 Protease Targeting Protease in "Super-Open" Conformation.

The Zika virus (ZIKV), a member of the Flaviviridae family, is considered a major health threat causing multiple cases of microcephaly in newborns and Guillain-Barré syndrome in adults. In this study, we targeted a transient, deep, and hydrophobic pocket of the "super-open" conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the active site pocket. After virtual docking screening of approximately seven million compounds against the novel allosteric site, we selected the top six candidates and assessed them in enzymatic assays. Six candidates inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations. These six compounds, targeting the selected protease pocket conserved in ZIKV, serve as unique drug candidates and open new opportunities for possible treatment against several flavivirus infections.

Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors.

Virtual screening is a widely used tool for drug discovery, but its predictive power can vary dramatically depending on how much structural data is available. In the best case, crystal structures of a ligand-bound protein can help find more potent ligands. However, virtual screens tend to be less predictive when only ligand-free crystal structures are available, and even less predictive if a homology model or other predicted structure must be used. Here, we explore the possibility that this situation can be improved by better accounting for protein dynamics, as simulations started from a single structure have a reasonable chance of sampling nearby structures that are more compatible with ligand binding. As a specific example, we consider the cancer drug target PPM1D/Wip1 phosphatase, a protein that lacks crystal structures. High-throughput screens have led to the discovery of several allosteric inhibitors of PPM1D, but their binding mode remains unknown. To enable further drug discovery efforts, we assessed the predictive power of an AlphaFold-predicted structure of PPM1D and a Markov state model (MSM) built from molecular dynamics simulations initiated from that structure. Our simulations reveal a cryptic pocket at the interface between two important structural elements, the flap and hinge regions. Using deep learning to predict the pose quality of each docked compound for the active site and cryptic pocket suggests that the inhibitors strongly prefer binding to the cryptic pocket, consistent with their allosteric effect. The predicted affinities for the dynamically uncovered cryptic pocket also recapitulate the relative potencies of the compounds (τb = 0.70) better than the predicted affinities for the static AlphaFold-predicted structure (τb = 0.42). Taken together, these results suggest that targeting the cryptic pocket is a good strategy for drugging PPM1D and, more generally, that conformations selected from simulation can improve virtual screening when limited structural data is available.

New 2‐Phenylquinoline‐Based Bis‐Methoxymethyl‐1,2,3‐Triazoles as Potential Antimicrobial Agents: Synthesis and Molecular Docking Studies against COVID‐19

AbstractSynthesis of novel 2‐phenylqinoline conjugated bis‐triazole analogues 8(a–n) demonstrated by a series of reactions concerning Vilsmeier‐Haack reaction, Suzuki‐Miyaura cross coupling, reduction of carbonyl groups, and click reaction. The structure of final compounds predicted based on IR, 1H, 13C NMR and Mass spectral data analysis. The title compounds were screened for their in vitro antimicrobial activities against two‐gram positive (S. aureus and S. pyogens) and two‐gram negative (P. aeruginosa and E. coli) bacteria, and two fungal strains (S. rolfs and F. ricini) by employing Amoxicillin and Bavistin as standard drugs, respectively. The compound containing 3‐chlorophenyl, 4‐(trifluoromethyl)phenyl, 3,4‐dimethylphenyl and 3‐fluoro‐4‐chlorophenyl residues, indicated promising antibacterial activity and the best antifungal activity was reported for compounds containing 3‐chlorophenyl, 4‐methoxyphenyl and 4‐(trifluoromethyl)phenyl residues. Further, Insilco screening of these compounds against COVID‐19 main protease of corona virus had displayed good docking scores and binding interactions in its active site pocket.

Insight into Tyrosine-Containing Pharmaceuticals as Potential Inhibitors of SARS-CoV-2 3CLpro and NSP16: Structural Analysis, Docking Studies, Molecular Dynamics Simulations, and Density Functional Theory Investigations

Tyrosine-containing pharmaceuticals’ (TPh) potential to inhibit SARS CoV-2 3-chymotrypsin-like proteases (3CLpro) and nonstructural protein 16 (NSP16) has been explored using docking studies, molecular dynamics simulations, and density functional theory. The TPh with FDA approval showed excellent contact with the active site pockets of 3CLpro and NSP16. Their binding affinity scores ranged from −5.8 to −4.9 kcal/mol and −6.3 to −4.8 for 3CLpro and NSP16, respectively. A 100-ns molecular dynamics simulation confirmed the stability of the carbidopa/NSP16 complex and N-acetyl tyrosine with both target enzymes. Further, the HOMO-LUMO transitions, molecular orbitals, and dipole moments of carbidopa, droxidopa, and N-acetyl tyrosine were computed using density functional theory (DFT). Considering N-acetyl tyrosine and carbidopa’s substantial inhibitory activity, it is recommended to investigate them further in order to explore their application for the treatment of COVID-19 or any other coronaviruses in the future.

Structural and enzymatic characterization of the sialidase SiaPG from Porphyromonas gingivalis.

The sialidases, which catalyze the hydrolysis of sialic acid from extracellular glycoconjugates, are a group of major virulence factors in various pathogenic bacteria. In Porphyromonas gingivalis, which causes human periodontal disease, sialidase contributes to bacterial pathogenesis via promoting the formation of biofilms and capsules, reducing the ability for macrophage clearance, and providing nutrients for bacterial colonization. Here, the crystal structure of the P. gingivalis sialidase SiaPG is reported at 2.1 Å resolution, revealing an N-terminal carbohydrate-binding domain followed by a canonical C-terminal catalytic domain. Simulation of the product sialic acid in the active-site pocket together with functional analysis enables clear identification of the key residues that are required for substrate binding and catalysis. Moreover, structural comparison with other sialidases reveals distinct features of the active-site pocket which might confer substrate specificity. These findings provide the structural basis for the further design and optimization of effective inhibitors to target SiaPG to fight against P. gingivalis-derived oral diseases.

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography.

Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5 Å resolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

A set of closely related methyltransferases for site-specific tailoring of anthraquinone pigments

Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdusluminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O-methyltransferases encoded in the genome of P.luminescens. Activity assays invitro and deletion experiments invivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found invitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation.

MicroED Structure of a Protoglobin Reactive Carbene Intermediate.

Microcrystal electron diffraction (MicroED) is an emerging technique that has shown great potential for describing new chemical and biological molecular structures. Several important structures of small molecules, natural products, and peptides have been determined using ab initio methods. However, only a couple of novel protein structures have thus far been derived by MicroED. Taking advantage of recent technological advances, including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of an Aeropyrum pernix protoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an α helix of ApePgb into a dynamic loop, making the catalytic active site more readily accessible. After exposing the tiny crystals to the substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineered ApePgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for the formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enable investigation of structures that was previously beyond reach.

Structural and molecular insights into tacrine-benzofuran hybrid induced inhibition of amyloid-β peptide aggregation and BACE1 activity

Amyloid-β (Aβ) aggregation and β-amyloid precursor protein cleaving enzyme 1 (BACE1) are the potential therapeutic drug targets for Alzheimer’s disease (AD). A recent study highlighted that tacrine-benzofuran hybrid C1 displayed anti-aggregation activity against Aβ42 peptide and inhibit BACE1 activity. However, the inhibition mechanism of C1 against Aβ42 aggregation and BACE1 activity remains unclear. Thus, molecular dynamics (MD) simulations of Aβ42 monomer and BACE1 with and without C1 were performed to inspect the inhibitory mechanism of C1 against Aβ42 aggregation and BACE1 activity. In addition, a ligand-based virtual screening followed by MD simulations was employed to explore potent new small-molecule dual inhibitors of Aβ42 aggregation and BACE1 activity. MD simulations highlighted that C1 promotes the non aggregating helical conformation in Aβ42 and destabilizes D23–K28 salt bridge that plays a vital role in the self-aggregation of Aβ42. C1 displays a favourable binding free energy (–50.7 ± 7.3 kcal/mol) with Aβ42 monomer and preferentially binds to the central hydrophobic core (CHC) residues. MD simulations highlighted that C1 strongly interacted with the BACE1 active site (Asp32 and Asp228) and active pockets. The scrutiny of interatomic distances among key residues of BACE1 highlighted the close flap (non-active) position in BACE1 on the incorporation of C1. The MD simulations explain the observed high inhibitory activity of C1 against Aβ aggregation and BACE1 in the in vitro studies. The ligand-based virtual screening followed by MD simulations identified CHEMBL2019027 (C2) as a promising dual inhibitor of Aβ42 aggregation and BACE1 activity. Communicated by Ramaswamy H. Sarma

In Silico and In Vitro Approach for Validating the Inhibition of Matrix Metalloproteinase-9 by Quercetin.

To validate the mechanism and inhibitory activity of quercetin against matrix metalloproteinase-9 (MMP-9) using a hybrid in silico and in vitro approach. The structure of MMP-9 was obtained from the Protein Data Bank, and the active site was identified using previous annotations from the Universal Protein Resource. The structure of quercetin was obtained from ZINC15. Molecular docking was performed to quantify the binding affinity of quercetin to the active site of MMP-9. The inhibitory effect of various concentrations of quercetin (0.0025, 0.025, 0.25, 1.0, and 1.5 mM) on MMP-9 was quantified using a commercially available fluorometric assay. The cytotoxicity of quercetin to immortalized human corneal epithelial cells (HCECs) was quantified by obtaining the metabolic activities of the cells exposed to various concentrations of quercetin for 24 hr. Quercetin interacts with MMP-9 by binding within the active site pocket and interacting with residues LEU 188, ALA 189, GLU 227, and MET 247. The binding affinity predicted by molecular docking was -9.9 kcal/mol. All concentrations of quercetin demonstrated significant inhibition of MMP-9 enzyme activity (all P <0.03). There was little to no reduction of HCEC metabolic activity after a 24-hr exposure to all concentrations of quercetin ( P >0.99). Quercetin inhibited MMP-9 in a dose-dependent manner and was well-tolerated by HCECs, suggesting a potential role in therapy for diseases with upregulated MMP-9 as part of its pathogenesis.

Identification of residues involved in allosteric signal transmission from amino acid binding site of pyruvate kinase muscle isoform 2.

PKM2 is a rate-limiting enzyme in the glycolytic process and is involved in regulating tumor proliferation. Several amino acids (AAs) such as Asn, Asp, Val, and Cys have been shown to bind to the AA binding pocket of PKM2 and modulate its oligomeric state, substrate binding affinity, and activity. Although previous studies have attributed that the main chain and side chain of bound AAs are responsible for initiating signal to regulate PKM2, the signal transduction pathway remains elusive. To identify the residues involved in signal transfer process, N70 and N75 located at two ends of a β strand connecting the active site and AA binding pocket were altered. Biochemical studies of these variants with various AA ligands (Asn, Asp, Val, and Cys), illustrate that N70 and N75, along with β1 connecting these residues are part of the signal transduction pathway between the AA binding pocket and the active site. The results demonstrate that mutation of N70 to D prevents the transfer of the inhibitory signal mediated by Val and Cys, whereas N75 to L alteration blocks the activating signal initiated by Asn and Asp. Taken together, this study confirms that N70 is one of the residues responsible for transmitting the inhibitory signal and N75 is involved in the activation signal flow.

Underlying Role of Hydrophobic Environments in Tuning Metal Elements for Efficient Enzyme Catalysis.

The catalytic functions of metalloenzymes are often strongly correlated with metal elements in the active sites. However, dioxygen-activating nonheme quercetin dioxygenases (QueD) are found with various first-row transition-metal ions when metal swapping inactivates their innate catalytic activity. To unveil the molecular basis of this seemingly promiscuous yet metal-specific enzyme, we transformed manganese-dependent QueD into a nickel-dependent enzyme by sequence- and structure-based directed evolution. Although the net effect of acquired mutations was primarily to rearrange hydrophobic residues in the active site pocket, biochemical, kinetic, X-ray crystallographic, spectroscopic, and computational studies suggest that these modifications in the secondary coordination spheres can adjust the electronic structure of the enzyme-substrate complex to counteract the effects induced by the metal substitution. These results explicitly demonstrate that such noncovalent interactions encrypt metal specificity in a finely modulated manner, revealing the underestimated chemical power of the hydrophobic sequence network in enzyme catalysis.

Anticancer, Antioxidant Activity and Molecular Docking Studies of Saccharumoside-B

Objectives: The present study reports antioxidant potential, the in-vitro antiproliferative activity of saccharumoside-B, and molecular docking studies on the binding affinity of saccharumoside-B towards various proteins involved in breast cancer pathogenesis. Methods: The in-vitro antioxidant activity of saccharumoside-B was determined by DPPH, superoxide and nitric oxide free radical scavenging assays. The antiproliferative activity was evaluated in-vitro by MTT assay. The binding affinity of saccharumoside-B towards the HSP90, HER2, human estrogen receptor and tyrosine-protein kinase C-SRC were determined by the molecular docking studies. Results: Saccharumoside-B showed a significant dose-dependent antioxidant activity and potent dose-dependent antiproliferative effect on the MCF-7 breast cancer cell line (IC50 = 22.57±0.39µM) among all other cell lines studied. Tamoxifen was used as a positive control for MCF-7 cell line (IC50 = 27.97±1.07µM). The IC50 of saccharumoside-B on normal MCF-10A cell line (IC50&gt;1000µM) showed a promising safety profile, whereas tamoxifen’s IC50 on MCF-10A normal cell line was found to be 29.6 ± 0.84 µM. The molecular docking analysis revealed that saccharumoside-B was inserted into the active site pockets of all the tested proteins involved in the breast cancer pathogenesis with varying binding affinities. Conclusion: The antioxidant studies revealed the potential antioxidant effect of saccharumoside-B. The in-vitro and in-silico studies are indicating the safe and multiple target protein inhibitory potential of saccharumoside-B against breast cancer. This study suggests that saccharumoside-B can be developed into a new class of anticancer drugs with a high safety profile in the future.

Novel 2-Thiouracil-5-Sulfonamide Derivatives: Design, Synthesis, Molecular Docking, and Biological Evaluation as Antioxidants with 15-LOX Inhibition.

New antioxidant agents are urgently required to combat oxidative stress, which is linked to the emergence of serious diseases. In an effort to discover potent antioxidant agents, a novel series of 2-thiouracil-5-sulfonamides (4-9) were designed and synthesized. In line with this approach, our target new compounds were prepared from methyl ketone derivative 3, which was used as a blocking unit for further synthesis of a novel series of chalcone derivatives 4a-d, thiosemicarbazone derivatives 5a-d, pyridine derivatives 6a-d and 7a-d, bromo acetyl derivative 8, and thiazole derivatives 9a-d. All compounds were evaluated as antioxidants against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2), lipid peroxidation, and 15-lipoxygenase (15-LOX) inhibition activity. Compounds 5c, 6d, 7d, 9b, 9c, and 9d demonstrated significant RSA in all three techniques in comparison with ascorbic acid and 15-LOX inhibitory effectiveness using quercetin as a standard. Molecular docking of compound 9b endorsed its proper binding at the active site pocket of the human 15-LOX which explains its potent antioxidant activity in comparison with standard ascorbic acid.

Roles of conserved active site residues in the IscS cysteine desulfurase reaction.

Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5'-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

Green synthesis of ferrocenyl chalcones against triple negative breast cancer

Without using any solvents, a green and highly efficient protocol for the preparation of ferrocenyl chalcones has been achieved by grinding between acetylferrocene and aldehyde. In addition, their in vitro antiproliferative activity against two breast cancer cell lines MCF-7 (estrogen receptor-positive) and MDA-MB-231 (triple negative breast cancer, TNBC) was also evaluated. Most of them exhibited excellent antiproliferative activity against MDA-MB-231 cells. The structure-activity relationship (SAR) study indicated that the aldehydes unit of ferrocenyl chalcones with halogens or dimethyl substitutions were favorable for anti-TNBC activity. Among them, compound 1d (IC50 = 2.4 ± 0.1 µM) exhibited the best anti-tumor effect on MDA-MB-231 cells. An enzymatic assay indicated that compound 1d (IC50 = 2.4 µM) was a potent COX-2 inhibitor. Molecular docking suggested 1d adopted an optimal binding pose in the COX-2 active-site pocket, and the carbonyl group of 1d formed the key hydrogen bond with the Y355 residue of COX-2

Targeted Degradation of Androgen Receptor by VNPP433-3β in Castration-Resistant Prostate Cancer Cells Implicates Interaction with E3 Ligase MDM2 Resulting in Ubiquitin-Proteasomal Degradation.

Targeted protein degradation is a fast-evolving therapeutic strategy to target even the traditionally undruggable target proteins. Contrary to the traditional small-molecule inhibitors of enzyme or receptor antagonists that bind the active site pockets in the target protein, molecular glue degraders facilitate interaction of target proteins with E3 ubiquitin ligases by stabilizing the ternary complex and induce physical proximity, thereby triggering ubiquitination and subsequent proteasomal degradation. AR plays a key role in all stages of prostate cancer. It is activated by the binding of androgenic hormones and transcriptionally regulates multiple genes including the ones that regulate cell cycle. Using HiBiT CRISPR cell line, biochemical methods, and RNA sequencing, we report the potential role of VNPP433-3β, the next generation galeterone analog as molecular glue that brings together AR, the key driver of prostate cancer and MDM2, an E3 ubiquitin ligase leading to ubiquitination and subsequent degradation of f-AR and AR-V7 in prostate cancer cells.

Insights into chlorogenic acids' efficient biosynthesis through Carthamus tinctorius cell suspension cultures and their potential mechanism as α-glucosidase inhibitors

Carthamus tinctorius is commonly used as food and medicine because it contains various bioactive components, particularly chlorogenic acids (CGAs). However, few research works focused on the development and application of C. tinctorius-derived CGAs. In this study, the technology system for efficient CGAs biosynthesis through C. tinctorius cells was successfully developed, and the feasibility of C. tinctorius cells-derived CGAs as α-glucosidase inhibitors was evaluated. At first, the inflorescence explant was used to induce C. tinctorius embryogenic callus (induction rate: 95%), in which a total of 10 CGAs, including rare caffeoyl and feruloyl-substituted derivatives, were identified and quantified by liquid chromatography-mass spectrometry. Secondly, an excellent C. tinctorius cell suspension culture was established. To further promote CGAs biosynthesis, parameters of elicitation combined with precursor feeding were optimized through Plackett-Burman’s Design and Central Composite Design. Under the optimal condition [cinnamic acid: 283.05 μM, methyl jasmonate: 80.98 μM and yeast extract: 666.66 mg·L−1], CGAs content could reach up to 103.234 mg·g−1 (DW), which was 2.55 times that of the cells in the initial condition. The CGAs production first time reached the highest level among plant-cultured cells in the available literature. At last, the inhibition activity test results indicated good inhibitory activities of C. tinctorius cells-derived CGAs against α-glucosidase (IC50: 0.15–1.38 g·L−1), especially di- and tri-acyl CGAs. Moreover, molecular docking simulation showed that these CGAs could interact with the active site pocket of α-glucosidase to thereby achieve the inhibitory activity. Therefore, these results provided a solid foundation for the large-scale production of CGAs and revealed the application prospects of CGAs in functional food and pharmaceutical industries as α-glucosidase inhibitor.