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Isolation, culture, and use of primary murine myoblasts in small-molecule screens.

ABSTRACT: Small-molecule screens (SMS) are often performed using transformed cell lines that have limited physiological relevance to the biological system being investigated, resulting in poor translational outcomes. To circumvent this limitation, we present a protocol to perform SMS in primary murine myoblasts. We describe steps for isolating primary skeletal muscle myoblasts with greater than 95% purity, then describe techniques to establish a robust dynamic range, and conclude with steps to initiate a successful SMS. For complete details on the use and execution of this protocol, please refer to Richler and Yaffe (1970),¹ Rando and Blau (1994),² and Earle et al. (2020).³.

SUBMITTER: Qu Y

PROVIDER: S-EPMC10025262 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Isolation, culture, and use of primary murine myoblasts in small-molecule screens.

Qu Yue Y Edwards Kaydine K Barrow Joeva J

STAR protocols 20230313 2

Small-molecule screens (SMS) are often performed using transformed cell lines that have limited physiological relevance to the biological system being investigated, resulting in poor translational outcomes. To circumvent this limitation, we present a protocol to perform SMS in primary murine myoblasts. We describe steps for isolating primary skeletal muscle myoblasts with greater than 95% purity, then describe techniques to establish a robust dynamic range, and conclude with steps to initiate a ...[more]

PMID: 36917603

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Project description:BackgroundLeishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.Methodology/principal findingsIn order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.Conclusions/significanceThe pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

Dataset Information

Isolation, culture, and use of primary murine myoblasts in small-molecule screens.

Publications

Isolation, culture, and use of primary murine myoblasts in small-molecule screens.

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