Investigations around RBM39 degrading arylsulfonamides

The cells in our body are a dynamic system that is constantly adapting to changing conditions. Their function is highly regulated by mechanisms, which go beyond the genome. The genes give the recipe to build the components in the system, but it is the molecular diversity that is introduced on the protein level which is crucial for the function. As such, proteins are the most diversified molecules in our body and have evolved complex networks that are involved in key cellular processes. Within these networks, they can either directly impact processes on a molecular level or indirectly do so by being part of a multi-protein complex. It is hence not surprising that protein misregulation or malfunction is at the bottom of a lot of diseases and is targeted in drug discovery.
Recently, the ubiquitin proteasomal pathway (UPP) has been manipulated to mediate targeted protein degradation (TPD) using small molecules. The development of such molecules has emerged from different independent angles, but it was the discovery of the mode-of-action of the clinically approved immunomodulatory drugs (IMiDs) that paved the way. They engage a UPP component (E3 ligase) and induce the ubiquitination and degradation of key proteins. The IMiDs and similar E3 binding molecules were used to develop chimeric molecules (PROTACs) which could degrade other proteins. In this work, we analyzed such a series of arysulfonamides (ArSulfs) that engage the E3 ligase CRL4DCAF15 reminiscent of the IMiDs and degrade the splicing factor RBM39.
In the first chapter, we look at PROTACs which were made to degrade the transcription elongation factor BRD4. The molecules entail the ArSulf Indisulam or derivatives thereof and the BRD4 inhibitor (+)JQ1. We show that, although the molecules can degrade BRD4 in a proteasomal-dependent manner, it is independent of the engagement with CRL4DCAF15. Our results show that degradation is CRL-dependent and suggest that it could be a general phenomenon when targeting BRD4 with (+)JQ1.
The following chapter summarizes our structural investigations around the RBM39-ArSulf-DCAF15 interaction. We conduct immunoprecipitation and pull-down experiments with the complex. A structure-activity relationship on the ArSulfs with a focus on Indisulam and E7820 helps us identify core elements of the scaffold and a site for modification. We use the site to make more PROTACs. The overall results indicate a very tight and specific interaction between RBM39-ArSulf-DCAF15 that offers little scope for a binary interaction with the ArSulfs in the absence of either protein. Hence, we made Indisulam derivatives with electrophilic warheads, which would engage in the complex but stay attached once RBM39 dissociates. The derivates were made using recently published structural data of the complex. We show the effects of the molecules in cells as well as purified proteins.
In the final chapter, we take a closer look at the effects of the ArSulfs in the cells. We look at what other proteins the ArSulfs can degrade but also investigate the other changes seen. We start by conducting tandem-mass tag (TMT) labelled proteomics experiments that help us identify other proteins affected by the treatment. By overlaying our proteomics data with RBM39 eCLIP data we identify splicing targets of RBM39 that show changes in protein levels upon RBM39 degradation. We validate these targets independently and hypothesize that the changes in protein levels come from aberrant splicing. Indeed, we demonstrate using transcript-specific qPCR that the relative level of transcript types can be correlated to the protein level change of RSRP1, as well as the kinesins KIF20A and KIF20B. A STRING network analysis with the significantly regulated proteins correlates with cell cycle and mitosis. Hence, we analyze the effect on the cell cycle with the ArSulfs to see if it can be attributed to the deregulation of the kinesins.