Schroeder, Martin, Thoma, Claudio, Liang, Xiaoyou, Hofmann, Andreas, Meili, Fabian, Thomas, Jason, Sigoillot, Frederic, Gleim, Scott, Vulpetti, Anna, Zoller, Thomas, Ferrand, Sandrine, Chung, BoYee, Andraos-Rey, Rita and Niederst, Matt (2023) Reinstating targeted protein degradation with DCAF1 PROTACs in CRBN PROTAC resistant settings. BioRxiv.

Abstract

Abstract to be written, for journal. Please find the synopsis for OAK reviewers only below:

In this publication we disclose the discovery of DCAF1 E3 ligase binding matter and the functionalization to PROTACs for TPD of BRD9, Tyrosine kinases (Dasatinib) and BTK. A lot of data has been initiated as part of the work by the ONC9LIG team (I was an integral part of). Project work on DCAF1 has been discontinued roughly 6-7 years ago (focus on BTK bifunctional degraders as ONC9BTK team). All compounds (IP inventory) have been shared with GDC in a previous email and the proposal to publish DCAF1 binders as well as the DCAF1-degraders was formally approved in August 2021 by the TPD steering committee including Jay and Karin.

We disclose 5 key messages:
• Discovery of DCAF1 as an essential ligase (DepMAP) and hypothesis to overcome CBRN mediated resistance. Leads into non-covalent DCAF1 binder discovery and characterisation, which can be functionalized for TPD for PROTACs (Figure 1)
• With a first degrader prototype we do show that endogenous mainly nuclear ligase DCAF1 can degrade BRD9 (BRD9-PROTAC, another nuclear protein. Key message is around in-depth chemical and genetic validation (Figure 2)
• We expand the degradation space with a second more promiscuous degrader prototype against tyrosine kinases (Dasatinib-DCAF1 PROTAC) and show that DCAF1 can even tackle cytoplasmic as well as membrane bound tyrosine kinases (Figure 3) – we touch here also on the tox observation
• We expand this further to an ONC target BTK with more specific PROTACs (linear and more complex linkers) that we characterize in-depth (Figure 4), here we describe a very systematic in vitro approach and show that unfortunately there is not a simple correlation between in vitro findings (complex, ubi etc.) and cellular degradation potency (as seen by many but not really worked up so systematically). Here we build up the path towards discovery of our most potent DCAF1-BTK degrader (JRF)
• JRF449 characterization and ultimate rescue experiment in CBRN-BTK PROTAC resistant cells where we do see BTK degradation and effect in proliferation with a DCAF1-BTK degrader even in CRBN resistant settings (in finalization) (draft Figure 5)
Key internal flags towards DCAF1 as an alternative ligase that led to termination were and still are:
• Discovery of only 1 chemical series (to be published in accompanying manuscript OAK 49705)
• This chemical series shows pan toxicity at >5uM (CLiP studies >300 cell lines tested revealed this)
o Rescue experiments to understand if this is on-target toxicity remained unsuccessful (Focused mutagenesis around donut-hole binding site, whole gene Variomics, endogenous gene editing around donut-hole locus)
o Until now tough we do not have a clear answer if this is off or on-target tox (DCAF1 is an essential ligase)
• We comment on these observations of tox briefly in the manuscript
• In addition, even tough we had extensive efforts on DCAF1-BTK degraders (50+ molecules) we never reached a potency range compared to CRBN-BTK (in-house) or (VHL, external). Our best degrader JRF449 (in publication has a DC50 of about 100nM)
o Literature also suggests that DCAF1 is somehow in a tetrameric self-inhibitory mode
We have had discussions with GDC colleagues concerning the chemical matter to be published in this manuscript and got verbal consent that the compounds can be disclosed. GDC decided not to patent the DCAF1 binders on their own and all the other elements of the PROTACs in this manuscript, thelinkers and war-heads are covered by own patents (BTK-binders and linkers) or in the public domain (BRD9 binder and Dasatinib).

Item Type:	Article
Keywords:	TPD, DCAF1
Date Deposited:	01 Sep 2023 00:45
Last Modified:	01 Sep 2023 00:45
URI:	https://oak.novartis.com/id/eprint/49742