Targeted Protein Degradation: From Chemical Biology to Drug Discovery

By controlling the stability, activity, or localization of crucial signaling molecules,
ubiquitylation plays an essential role for metazoan development and its aberrant function
is often linked to disease. A well-established function of ubiquitylation is in mediating
robust stress signaling that allows organisms to detect and ameliorate adverse conditions
and thereby maintain cellular integrity. Here, I will present genetic screens that allowed us
to identify a novel ubiquitin-dependent stress response network with essential functions in
metazoan tissue development. By dissecting the requirements for substrate recognition, we
were able to develop new approaches to modulate the activity of an essential E3 ligase by
small molecules. Our work illustrates how stress signaling can be used to ensure robust
stem cell differentiation, and it opens the door to interesting new therapeutic approaches
against a set of pediatric or metabolic diseases.

Treatment of relapsed or refractory lymphoid neoplasms is a significant clinical challenge. Venetoclax is a selective BCL-2 inhibitor that induces apoptosis and is efficacious in chronic lymphocytic leukemia; however, single agent efficacy in other lymphoid neoplasms is limited. Here we report a mechanismdriven combination strategy for maximizing anti-tumor responses in relapsed or refractory non-Hodgkin lymphoma. The antibody-drug conjugate polatuzumab vedotin promotes degradation of the pro-survival BCL-2 family member MCL-1 and causes durable tumor regressions in relapsed or refractory nonHodgkin lymphoma models when combined with venetoclax and the anti-CD20 antibody obinutuzumab. Targeting both BCL-2 and MCL-1 with venetoclax and polatuzumab vedotin is a rationally-designed therapeutic regimen that overcomes treatment resistance and has provided early promising disease responses in a Phase 1b clinical trial for relapsed or refractory non-Hodgkin lymphoma.

Coauthors: Dhara N. Amin[1,2], Raghuveer Singh Mali[3], Jason Oeh, PhD[3], Anuradha Zindal[2], Ellen Rae-Ingalla[3], Shang-Fan Yu[3], Lisa Musick[4], Jamie Hirata[4], Mehrdad Mobasher[4], Rajat Bannerji[5], Maxwell M. Krem[6], Elisabeth Lasater[3], Andrew G. Polson[3], Deepak Sampath[3], and Christopher R. Flowers[7].

1. Department of Discovery Oncology, Genentech, South San Francisco.
2. Department of Early Discovery Biochemistry, Genentech, South San Francisco.
3. Department of Translational Oncology, Genentech, South San Francisco.
4. Product Development Oncology, Genentech, South San Francisco.
5. Rutgers Cancer Institute of New Jersey and Robert Wood Johnson Medical School, 195 Little Albany
Street, New Brunswick, NJ.
6. Division of Blood and Marrow Transplantation, Department of Medicine, James Graham Brown Cancer
Center, University of Louisville School of Medicine.
7. Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of
Medicine, Atlanta.

Cereblon modulators are small molecules that bind to the protein cereblon, and include the clinically
approved drugs thalidomide, lenalidomide and pomalidomide. Cereblon is part of the CRL4-CRBN E3
ubiquitin ligase complex that catalyzes the transfer of ubiquitin to mark target proteins for degradation.
Cereblon modulating drugs bind to the surface of cereblon to form a ‘hotspot’ for protein-protein
interactions triggering the recruitment of proteins to the ubiquitin ligase complex. Upon binding, target
proteins can be ubiquitinated and subsequently degraded by the proteasome. A search for cereblon
modulators that degrade new proteins lead to the discovery of CC-885, which exhibits potent anti-tumor
activity. The potency of CC-885 is caused by the degradation of the protein translation factor GSPT1.

A crystal structure of cereblon in complex with CC-885 and GSPT1 reveals that GSPT1 interacts with the
surface of cereblon via a molecular feature, or ‘degron’, that is conserved amongst unrelated cereblon
substrates. The degron does not require a canonical small molecule binding site on the substrate protein
and can be found in ‘undruggable’ protein families. A search for the degron in the C2H2 zinc finger
protein family revealed that the embryonic transcription factor SALL4 is a thalidomide-dependent cereblon
substrate. Mutations in SALL4 cause syndromes with a clinical presentation so close to that of
thalidomide embryopathy that they have been misdiagnosed. Thus, we believe we have uncovered a
major molecular mechanism for thalidomide teratogenicity.

This talk will describe our continuing efforts to identify new substrates and activities for cereblon
modulators.

The degradation of ubiquitin-tagged proteins by the 26S proteasome is a highly complex process with several well-coordinated sub-steps, including ubiquitin recognition, substrate engagement, unfolding, deubiquitination, translocation, and proteolytic cleavage. Our recently developed FRET- and fluorescence-based assays, relying on the labeling of proteasome-incorporated unnatural amino acids, allowed us to dissect the kinetics and coordination of individual processing steps, and study the coupled conformational changes of the proteasome. Single-molecule measurements thereby revealed exciting details about the dynamics of the proteasome and the progression of protein substrates through the different stages of degradation. By using substrates with various architectures, we gained a better understanding of how a substrate’s geometry and ubiquitin modifications affect the degradation rate and which processing steps are rate-limiting. We found that the first ubiquitin chain on a substrate is removed more slowly than chains subsequently arriving at the entrance to the proteasome pore, and that additional ubiquitin chains obstructing a substrate’s flexible initiation region are cleaved off in a slow, translocation-independent manner that is rate-limiting for degradation. Furthermore, our studies revealed that the attachment of ubiquitins to lysines on folded domains can significantly reduce the thermodynamic stability and make substrates amenable to proteasomal degradation even in the absence of a flexible initiation region. Well-folded ubiquitinated proteins are also assumed to be partially or completely unfolded by Cdc48/p97 for subsequent degradation by the proteasome, and using a minimal in vitro-reconstituted system we recently showed how these molecular machines collaborate in processing substrates lacking unstructured regions.  Our data thus provide important new insight into how the proteasome may prioritize its substrates in the cell, how degradation is affected by various substrate characteristics, and how Cdc48/p97 prepares substrates for degradation.

Small molecules that induce protein degradation through ligase-mediated ubiquitination have shown
considerable promise as a new pharmacological modality with thalidomide and related IMiDs providing
the clinical proof of concept for this novel approach. While significant progress has been made towards
chemically induced targeted protein degradation using heterobifunctional small molecule ligands (often
referred to as degraders or PROTACs), such molecules still necessitate a high affinity ligand for their
target. Molecular glues, akin to thalidomide circumvent the need for such a ligand moiety, however,
many of the fundamental principles governing neo-substrate recruitment remain elusive. We will
present recent work towards a better understanding of the molecular principles that govern neosubstrate recruitment by small molecules akin to thalidomide, and its application to the development
of small molecule degraders.

Coauthors: Tyler Faust,  Hojong Yoon, Radoslaw P. Nowak,  and Katherine A. Donovan; Harvard Medical School and Dana-Farber Cancer Institute, Boston.

Characterizing and optimizing PROTACs for degradation efficacy represents a significant challenge,
particularly in understanding processes and potential failure points that control whether degradation will
result. Currently, the availability of live cell assays to interrogate the multiple steps that are required to
achieve degradation by PROTACs is severely lacking. Here, we present a live-cell, luminescence-based
technology platform that enables characterization of PROTAC compound mechanism of action using
either ectopic or endogenous target expression formats. We employ CRISPR/Cas9 endogenous tagging
of target proteins with the small peptide, HiBiT, which has high affinity for and can complement with the
LgBiT protein to produce NanoBiT luminescence. This allows for sensitive detection of endogenous
protein levels in living cells, and can also serve as a BRET energy donor to study protein:protein or
protein:small molecule interactions. Using this combinatorial approach, we demonstrate the ability to
measure permeability effects and binding affinities of PROTAC compounds to both target and E3 ligase,
as well as monitor the kinetics of the subsequent ternary complex (target:PROTAC:E3 ligase) formation,
target ubiquitination and recruitment to the proteasome in live cells. We further show the power of this
technology in extended kinetic monitoring of endogenous target protein levels and the ability to quantify
degradation rates, maximal levels of degradation, potency, persistence of degradation, protein recovery,
and how these parameters are closely coupled to the specific mechanism of action. These studies
facilitate discernment of individual parameters required for successful degradation, ultimately enabling
chemical design strategies for optimization and rank ordering of therapeutic PROTAC compounds.

The proteolysis targeting chimera (PROTAC) concept was first introduced in 2001 with
the objective of induction of selective target protein degradation by hijacking the cellular
E3 ubiquitination ligase systems. The PROTAC strategy has recently gained momentum
for its promising potential for the discovery of completely new classes of medicines. In
this talk, I will present our most recent progress in the design and discovery of PROTAC
degraders for a classical undruggable target, the signal transducer and activator of
transcription 3 (STAT3). I will also discuss our extensive in vitro and in vivo studies of our
potent and highly selective PROTAC STAT3 degraders and their therapeutic potential. I
will highlight the key differences between inhibitors and degraders of STAT3 in vitro and
in vivo.

Targeted Protein Degradation is an emerging therapeutic modality that utilizes bifunctional molecules, whereby one end binds to the Protein Of Interest (POI) while the other hijacks cellular quality control mechanisms to induce the degradation of the target protein, rather than inhibiting his function.

This presentation will highlight internal efforts to build state-of the art assay cascades towards understanding the molecular mechanism underlying this intriguing biology, the build of a proteomics platform to define the binding and degradation selectivity of protein degraders and the hit finding for novel E3 ligases ligands. The identification of novel degraders against two Oncogenic targets (BCL6 and EED) and their utility as target validation tools and as potential therapeutics for many solid and hematological malignancies will be described.

Lenalidomide, a derivative of thalidomide, is a highly effective drug for the treatment of myelodysplastic syndrome (MDS) with del(5q), multiple myeloma, and some additional B cell lymphomas. Lenalidomide directly binds the CRL4-CRBN E3 ubiquitin ligase. In multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 and IKZF3, to the CRBN substrate adapter; increases the ubiquitination of these substrates; and causes the targeted degradation of IKZF1 and IKZF3, transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells. We identified and characterized a degron sequence that mediates binding of substrates to the ubiquitin ligase and confers lenalidomideinduced degradation onto reporters. IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation, and we found that genetic inactivation of IKZF1 or IKZF3 in lenalidomidesensitive multiple myeloma cells causes severe growth inhibition. In del(5q) MDS, lenalidomide targets CSNK1A1 for destruction. Haploinsufficiency for CSNK1A1, a gene located within the common deleted region on chromosome 5q, results in selective targeting of the del(5q) clone. CSNK1A1 is essential for the survival of AML cells as well and may therefore provide a mechanism of action for lenalidomide in AML as well, though with a decreased therapeutic index compared with the use of lenalidomide in del(5q) MDS. Different lenalidomide analogues induce degradation of distinct sets of substrates, indicating that novel analogues may have novel clinical activities.

The increasingly atomic resolution of human biology presents an unprecedented opportunity for the innovation of definitive medicines for life-threatening diseases.  Still, many well-defined, high-value protein targets remain beyond the limits of historical efforts in ligand discovery.  Often challenging the discovery of therapeutics that disable intracellular proteins is the real or perceived limitation of small molecules to engage non-canonical protein folds and disrupt a biophysical or biochemical function.  We have therefore undertaken to invest in the discipline of chemical biology as an organizing principle in drug discovery, so as to create vast numbers and new types of small molecules to advance the science of therapeutics.  In this seminar, I will discuss progress in the field of targeted protein degradation, inspired by natural products and advances in chemical-induced dimerization.  I will highlight both foundational research as well as our recent, first all-chemical approach that establishes design principles for new ligands, demonstrates extensibility to numerous cellular targets and informs mechanistic cellular biology.  Importantly, lead optimization of chemical probes brings extraordinary (sub-nanomolar) potency, reveals catalyst-like activity, and now emanates first-in-class therapeutics.  Finally, I will discuss learnings from our Targeted Protein Degradation Initiative, including curious challenges around which our ongoing research organizes.