Dataset Information

Sulphated penta-galloyl glucopyranoside (SPGG) is glycosaminoglycan mimetic allosteric inhibitor of cathepsin G.

ABSTRACT:

Objective

Cathepsin G (CatG) is a cationic serine protease with wide substrate specificity. CatG is reported to play a role in several inflammatory pathologies. Thus, we aimed at identifying a potent and allosteric inhibitor of CatG to be used as a platform in further drug development opportunities.

Methods

Chromogenic substrate hydrolysis assays were used to evaluate the inhibition potency and selectivity of SPGG towards CatG. Salt-dependent studies, Michaelis-Menten kinetics and SDS-PAGE were exploited to decipher the mechanism of CatG inhibition by SPGG. Molecular modelling was also used to identify a plausible binding site.

Key findings

SPGG displayed an inhibition potency of 57 nM against CatG, which was substantially selective over other proteases. SPGG protected fibronectin and laminin against CatG-mediated degradation. SPGG reduced V_MAX of CatG hydrolysis of a chromogenic substrate without affecting K_M, suggesting an allosteric mechanism. Resolution of energy contributions indicated that non-ionic interactions contribute ~91% of binding energy, suggesting a substantial possibility of specific recognition. Molecular modelling indicated that SPGG plausibly binds to an anion-binding sequence of ¹⁰⁹SRRVRRNRN¹¹⁷.

Conclusion

We present the discovery of SPGG as the first small molecule, potent, allosteric glycosaminoglycan mimetic inhibitor of CatG. SPGG is expected to open a major route to clinically relevant allosteric CatG anti-inflammatory agents.

SUBMITTER: Al-Horani RA

PROVIDER: S-EPMC9942669 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Publications

Sulphated penta-galloyl glucopyranoside (SPGG) is glycosaminoglycan mimetic allosteric inhibitor of cathepsin G.

Al-Horani Rami A RA Afosah Daniel K DK Kar Srabani S Aliter Kholoud F KF Mottamal Madhusoodanan M

RPS pharmacy and pharmacology reports 20230106 1

<h4>Objective</h4>Cathepsin G (CatG) is a cationic serine protease with wide substrate specificity. CatG is reported to play a role in several inflammatory pathologies. Thus, we aimed at identifying a potent and allosteric inhibitor of CatG to be used as a platform in further drug development opportunities.<h4>Methods</h4>Chromogenic substrate hydrolysis assays were used to evaluate the inhibition potency and selectivity of SPGG towards CatG. Salt-dependent studies, Michaelis-Menten kinetics and ...[more]

PMID: 36844783

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Project description:BackgroundLimited options for the treatment of cartilage damage have driven the development of tissue engineered or cell therapy alternatives reliant on ex vivo cell expansion. The study of chondrogenesis in primary cells is difficult due to progressive cellular aging and senescence. Immortalisation via the reintroduction of the catalytic component of telomerase, hTERT, could allow repeated, longitudinal studies to be performed while bypassing senescent phenotypes.MethodsThree human cell types: bone marrow-derived stromal cells (BMA13), embryonic stem cell-derived (1C6) and chondrocytes (OK3) were transduced with hTERT (BMA13H, 1C6H and OK3H) and proliferation, surface marker expression and tri-lineage differentiation capacity determined. The sulphated glycosaminoglycan (sGAG) content of the monolayer and spent media was quantified in maintenance media (MM) and pro-chondrogenic media (PChM) and normalised to DNA.ResultshTERT expression was confirmed in transduced cells with proliferation enhancement in 1C6H and OK3H cells but not BMA13H. All cells were negative for leukocyte markers (CD19, CD34, CD45) and CD73 positive. CD14 was expressed at low levels on OK3 and OK3H and HLA-DR on BMA13 (84.8%). CD90 was high for BMA13 (84.9%) and OK3 (97.3%) and moderate for 1C6 (56.7%), expression was reduced in BMA13H (33.7%) and 1C6H (1.6%). CD105 levels varied (BMA13 87.7%, 1C6 8.2%, OK3 43.3%) and underwent reduction in OK3H (25.1%). 1C6 and BMA13 demonstrated osteogenic and adipogenic differentiation but mineralised matrix and lipid accumulation appeared reduced post hTERT transduction. Chondrogenic differentiation resulted in increased monolayer-associated sGAG in all primary cells and 1C6H (p<0.001), and BMA13H (p<0.05). In contrast OK3H demonstrated reduced monolayer-associated sGAG in PChM (p<0.001). Media-associated sGAG accounted for ?55% (PChM-1C6) and ?74% (MM-1C6H).ConclusionIn conclusion, hTERT transduction could, but did not always, prevent senescence and cell phenotype, including differentiation potential, was affected in a variable manner. As such, these cells are not a direct substitute for primary cells in cartilage regeneration research.

Dataset Information

Sulphated penta-galloyl glucopyranoside (SPGG) is glycosaminoglycan mimetic allosteric inhibitor of cathepsin G.

Objective

Methods

Key findings

Conclusion

Publications

Sulphated penta-galloyl glucopyranoside (SPGG) is glycosaminoglycan mimetic allosteric inhibitor of cathepsin G.

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