Towards Small Molecules that Induce Expression of Reprogramming Factor OCT4

Reprogramming involves inducing expression of reprogramming factors, canonically OSKM (OCT4, SOX2, KLF4, and MYC), the Yamanaka factors. When expressed for a sufficiently long time, a period of days to weeks, some fraction of OSKM-expressing cells dedifferentiate into induced pluripotent stem cells (iPSCs). Before that happens, however, beneficial epigenetic changes occur, resetting a cell to a more youthful pattern of gene expression, resulting in improvements such as restored mitophagy and mitochondrial function. The present focus of the industry is to find a way to safely apply transient exposure to reprogramming factors as a therapy, producing epigenetic rejuvenation without dedifferentiation. Initial results in animal models are promising, but a great deal of work yet lies ahead.

Because the bulk of medical research and development is focused on small molecule development rather than gene therapy, the excitement surrounding therapeutic reprogramming as a whole translates to a strong interest in finding ways to use small molecules to induce reprogramming factor expression. This idea that reprogramming can be applied directly as a therapy, rather than being a way to produce iPSCs for regenerative medicine, is still in the comparatively early stages, however. There is a great deal of funding, and new information is arriving at a fast pace, but it still takes time to make meaningful progress.

Development of a next-generation endogenous OCT4 inducer and its anti-aging effect in vivo

Octamer-binding transcription factor 4 (OCT4) is a member of the POU transcription factor family and functions as one of the master regulators in initiating reprogramming and maintaining pluripotency. Independent groups have observed rejuvenation in partially reprogrammed mice, characterized by the reversal of aging marks and improved tissue regeneration. Consequently, chemicals capable of (partially) activating endogenous reprogramming-associated transcription factors hold promise as potential candidates in anti-aging therapy.

Several studies have reported small molecule inducers of endogenous OCT4, including forskolin (a cAMP agonist), and pyrrolo[2,3-b]pyridine based OCT-activating compound 1 (OAC1). However, to date, a small molecule capable of completely replacing or reactivating OCT4 or its analogues in the reprogramming of somatic cells, has not yet been reported.

Recently, we conducted high-throughput screening and identified a series of compounds called OCT4-inducing compounds (O4Is). These compounds have the ability to sustain the maintenance of human iPSCs by promoting the expression of endogenous OCT4, including 4-(benzyloxy)phenyl derivatives (O4I1s), 2-aminothiazoles (O4I2s) and imidazopyrimidines (O4I3s).

In this work, analysis of cellular metabolic products revealed that hydrolysis, especially in pluripotent cells, ablated the activity of O4I2-ester derivatives, which led us to design a second generation of O4I2 derivatives with improved metabolic stability, including a compound called O4I4. By combining O4I4 with the ectopic expression of SOX2, KLF4, L-MYC and LIN28 (collectively designated as "CSKML"), we successfully reprogrammed human fibroblasts into iPSCs. In C. elegans and Drosophila O4I4 extended their lifespans, suggesting the potential application of O4I4 and other reprogramming-associated chemicals in regenerative medicine and anti-aging therapy.

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