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1.
Bioorg Med Chem ; 102: 117658, 2024 Mar 15.
Article in English | MEDLINE | ID: mdl-38460487

ABSTRACT

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 µM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure-activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.


Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Cell Cycle Proteins , Aurora Kinase A , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , DNA Replication , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use
2.
Exp Mol Med ; 56(4): 827-835, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38556551

ABSTRACT

Intracellular retrograde transport in eukaryotic cells relies exclusively on the molecular motor cytoplasmic dynein 1. Unlike its counterpart, kinesin, dynein has a single isoform, which raises questions about its cargo specificity and regulatory mechanisms. The precision of dynein-mediated cargo transport is governed by a multitude of factors, including temperature, phosphorylation, the microtubule track, and interactions with a family of activating adaptor proteins. Activating adaptors are of particular importance because they not only activate the unidirectional motility of the motor but also connect a diverse array of cargoes with the dynein motor. Therefore, it is unsurprising that dysregulation of the dynein-activating adaptor transport machinery can lead to diseases such as spinal muscular atrophy, lower extremity, and dominant. Here, we discuss dynein motor motility within cells and in in vitro, and we present several methodologies employed to track the motion of the motor. We highlight several newly identified activating adaptors and their roles in regulating dynein. Finally, we explore the potential therapeutic applications of manipulating dynein transport to address diseases linked to dynein malfunction.


Subject(s)
Cytoplasmic Dyneins , Humans , Cytoplasmic Dyneins/metabolism , Animals , Biological Transport , Microtubules/metabolism , Dyneins/metabolism
3.
Adv Mater ; 36(19): e2308837, 2024 May.
Article in English | MEDLINE | ID: mdl-38351715

ABSTRACT

As large molecular tertiary structures, some proteins can act as small robots that find, bind, and chaperone target protein clients, showing the potential to serve as smart building blocks in self-assembly fields. Instead of using such intrinsic functions, most self-assembly methodologies for proteins aim for de novo-designed structures with accurate geometric assemblies, which can limit procedural flexibility. Here, a strategy enabling polymorphic clustering of quaternary proteins, exhibiting simplicity and flexibility of self-assembling paths for proteins in forming monodisperse quaternary cage particles is presented. It is proposed that the enzyme protomer DegQ, previously solved at low resolution, may potentially be usable as a threefold symmetric building block, which can form polyhedral cages incorporated by the chaperone action of DegQ in the presence of protein clients. To obtain highly monodisperse cage particles, soft, and hence, less resistive client proteins, which can program the inherent chaperone activity of DegQ to efficient formations of polymorphic cages, depending on the size of clients are utilized. By reconstructing the atomic resolution cryogenic electron microscopy DegQ structures using obtained 12- and 24-meric clusters, the polymorphic clustering of DegQ enzymes is validated in terms of soft and rigid domains, which will provide effective routes for protein self-assemblies with procedural flexibility.


Subject(s)
Protein Structure, Quaternary , Serine Endopeptidases , Cryoelectron Microscopy , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism
4.
Int J Biol Sci ; 19(16): 5245-5256, 2023.
Article in English | MEDLINE | ID: mdl-37928271

ABSTRACT

Adipocytes are adipose tissues that supply energy to the body through lipids. The two main types of adipocytes comprise white adipocytes (WAT) that store energy, and brown adipocytes (BAT), which generate heat by burning stored fat (thermogenesis). Emerging evidence indicates that dysregulated adipocyte senescence may disrupt metabolic homeostasis, leading to various diseases and aging. Adipocytes undergo senescence via irreversible cell-cycle arrest in response to DNA damage, oxidative stress, telomere dysfunction, or adipocyte over-expansion upon chronic lipid accumulation. The amount of detectable BAT decreases with age. Activation of cell cycle regulators and dysregulation of adipogenesis-regulating factors may constitute a molecular mechanism that accelerates adipocyte senescence. To better understand the regulation of adipocyte senescence, the effects of post-translational modifications (PTMs), is essential for clarifying the activity and stability of these proteins. PTMs are covalent enzymatic protein modifications introduced following protein biosynthesis, such as phosphorylation, acetylation, ubiquitination, or glycosylation. Determining the contribution of PTMs to adipocyte senescence may identify new therapeutic targets for the regulation of adipocyte senescence. In this review, we discuss a conceptual case in which PTMs regulate adipocyte senescence and explain the mechanisms underlying protein regulation, which may lead to the development of effective strategies to combat metabolic diseases.


Subject(s)
Adipocytes, Brown , Adipose Tissue, Brown , Adipose Tissue, Brown/metabolism , Adipocytes, Brown/metabolism , Adipogenesis/genetics , Protein Processing, Post-Translational , Cell Cycle Checkpoints
5.
Sci Adv ; 9(16): eadf8582, 2023 04 21.
Article in English | MEDLINE | ID: mdl-37083534

ABSTRACT

Aurora kinase A (AURKA) performs critical functions in mitosis. Thus, the activity and subcellular localization of AURKA are tightly regulated and depend on diverse factors including interactions with the multiple binding cofactors. How these different cofactors regulate AURKA to elicit different levels of activity at distinct subcellular locations and times is poorly understood. Here, we identified a conserved region of CEP192, the major cofactor of AURKA, that mediates the interaction with AURKA. Quantitative binding studies were performed to map the interactions of a conserved helix (Helix-1) within CEP192. The crystal structure of Helix-1 bound to AURKA revealed a distinct binding site that is different from other cofactor proteins such as TPX2. Inhibiting the interaction between Helix-1 and AURKA in cells led to the mitotic defects, demonstrating the importance of the interaction. Collectively, we revealed a structural basis for the CEP192-mediated AURKA regulation at the centrosome, which is distinct from TPX2-mediated regulation on the spindle microtubule.


Subject(s)
Aurora Kinase A , Spindle Apparatus , Aurora Kinase A/genetics , Aurora Kinase A/metabolism , Spindle Apparatus/metabolism , Centrosome/metabolism , Microtubules/metabolism , Mitosis
6.
Aquat Toxicol ; 260: 106573, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37210931

ABSTRACT

In this study, we aimed to identify novel compounds that could afford protection against cisplatin-induced ototoxicity by employing both cell- and zebrafish (Danio rerio)-based screening platforms. We screened 923 US Food and Drug Administration-approved drugs to identify potential compounds exhibiting protective effects against cisplatin-induced ototoxicity in HEI-OC1 cells (auditory hair cell line). The screening strategy identified esomeprazole and dexlansoprazole as the primary hit compounds. Subsequently, we examined the effects of these compounds on cell viability and apoptosis. Our results revealed that esomeprazole and dexlansoprazole inhibited organic cation transporter 2 (OCT2), thus providing in vitro evidence that these compounds could ameliorate cisplatin-induced ototoxicity by directly inhibiting OCT2-mediated cisplatin transport. In vivo, the protective effects were validated using zebrafish; esomeprazole was found to decrease cisplatin-induced hair cell damage in neuromasts. Furthermore, the esomeprazole-treated group showed a significantly lower number of TUNEL-positive cells than the cisplatin-treated group. Collectively, our findings revealed that esomeprazole exerts a protective effect against cisplatin-induced hair cell damage in both HEI-OC1 cells and a zebrafish model.


Subject(s)
Antineoplastic Agents , Ototoxicity , Water Pollutants, Chemical , Animals , Cisplatin/toxicity , Antineoplastic Agents/toxicity , Zebrafish/metabolism , Esomeprazole/pharmacology , Dexlansoprazole/pharmacology , Cell Line , Reactive Oxygen Species/metabolism , Water Pollutants, Chemical/toxicity , Apoptosis , Cell Survival
7.
Acta Crystallogr D Struct Biol ; 78(Pt 4): 424-434, 2022 Apr 01.
Article in English | MEDLINE | ID: mdl-35362466

ABSTRACT

D-Alanylation of the teichoic acids of the Gram-positive bacterial cell wall plays crucial roles in bacterial physiology and virulence. Deprivation of D-alanine from the teichoic acids of Staphylococcus aureus impairs biofilm and colony formation, induces autolysis and ultimately renders methicillin-resistant S. aureus highly susceptible to antimicrobial agents and host defense peptides. Hence, the D-alanylation pathway has emerged as a promising antibacterial target against drug-resistant S. aureus. D-Alanylation of teichoic acids is mediated via the action of four proteins encoded by the dlt operon, DltABCD, all four of which are essential for the process. In order to develop novel antimicrobial agents against S. aureus, the D-alanyl carrier protein ligase DltA, which is the first protein in the D-alanylation pathway, was focused on. Here, the crystal structure of DltA from the methicillin-resistant S. aureus strain Mu50 is presented, which reveals the unique molecular details of the catalytic center and the role of the P-loop. Kinetic analysis shows that the enantioselectivity of S. aureus DltA is much higher than that of DltA from other species. In the presence of DltC, the enzymatic activity of DltA is increased by an order of magnitude, suggesting a new exploitable binding pocket. This discovery may pave the way for a new generation of treatments for drug-resistant S. aureus.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , Staphylococcus aureus , Bacterial Proteins/chemistry , Carrier Proteins/metabolism , Kinetics , Ligases , Methicillin-Resistant Staphylococcus aureus/metabolism
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