A Shift in Thinking: Cellular Thermal Shift Assay-Enabled Drug Discovery.

A decade has passed since the cellular thermal shift assay (CETSA) was introduced to the drug discovery community. Over the years, the method has guided numerous projects by providing insights about, for example, target engagement, lead generation, target identification, lead optimization, and preclinical profiling. With this Microperspective, we intend to highlight recently published applications of CETSA and how the data generated can enable efficient decision-making and prioritization throughout the drug discovery and development value chain.

A Tale of Two Tails: Efficient Profiling of Protein Degraders by Specific Functional and Target Engagement Readouts.

Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance, and reaching "undruggable" proteins compared with traditional small-molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation; instead, one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs), understanding the binding characteristics of the PoI alone is not sufficient.

Pax6 is required intrinsically by thalamic progenitors for the normal molecular patterning of thalamic neurons but not the growth and guidance of their axons.

Background: In mouse embryos, the Pax6 transcription factor is expressed in the progenitors of thalamic neurons but not in thalamic neurons themselves. Its null-mutation causes early mis-patterning of thalamic progenitors. It is known that thalamic neurons generated by Pax6 (-/-) progenitors do not develop their normal connections with the cortex, but it is not clear why.

Cell-autonomous repression of Shh by transcription factor Pax6 regulates diencephalic patterning by controlling the central diencephalic organizer.

During development, region-specific patterns of regulatory gene expression are controlled by signaling centers that release morphogens providing positional information to surrounding cells. Regulation of signaling centers themselves is therefore critical. The size and the influence of a Shh-producing forebrain organizer, the zona limitans intrathalamica (ZLI), are limited by Pax6.

MBD3/NuRD facilitates induction of pluripotency in a context-dependent manner.

The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for embryonic development and pluripotent stem cell differentiation. In this study, we investigated whether NuRD is also involved in the reverse biological process of induction of pluripotency in neural stem cells. By knocking out MBD3, an essential scaffold subunit of the NuRD complex, at different time points in reprogramming, we found that efficient formation of reprogramming intermediates and induced pluripotent stem cells from neural stem cells requires NuRD activity.

The NuRD component Mbd3 is required for pluripotency of embryonic stem cells.

Cells of early mammalian embryos have the potential to develop into any adult cell type, and are thus said to be pluripotent. Pluripotency is lost during embryogenesis as cells commit to specific developmental pathways. Although restriction of developmental potential is often associated with repression of inappropriate genetic programmes, the role of epigenetic silencing during early lineage commitment remains undefined.

Kaiso-deficient mice show resistance to intestinal cancer.

Kaiso is a BTB domain protein that associates with the signaling molecule p120-catenin and binds to the methylated sequence mCGmCG or the nonmethylated sequence CTGCNA to modulate transcription. In Xenopus laevis, xKaiso deficiency leads to embryonic death accompanied by premature gene activation in blastulae and upregulation of the xWnt11 gene. Kaiso has also been proposed to play an essential role in mammalian synapse-specific transcription.

MeCP2 in neurons: closing in on the causes of Rett syndrome.

The discovery in 1999 that Rett syndrome (RTT) is caused by mutations in a gene encoding the methyl-CpG-binding repressor protein MECP2 provided a significant breakthrough in the understanding of this devastating disease. The subsequent production of Mecp2 knockout mice 2 years later provided an experimental resource to better understand how mutations in the MECP2 gene result in RTT. This paper reviews the recent progress in understanding when and where MeCP2 function becomes important in the developing brain, why MeCP2 protein levels are crucial, which genes are normally silenced by MeCP2, and how misexpression of these targets might lead to the clinical manifestations of RTT.