A phenotypic screen with Trypanosoma brucei for discovering small molecules that target the SLiM-binding pocket of proliferating cell nuclear antigen orthologs.

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric protein complex that clamps around DNA to tether DNA polymerases to the template during replication and serves as a hub for many other interacting proteins. It regulates DNA metabolic processes and other vital cellar functions through the binding of proteins having short linear motifs (SLiMs) like the PIP-box (PCNA-interacting protein-box) or the APIM (AlkB homolog 2 PCNA-interacting motif) in the hydrophobic pocket where SLiMs bind. However, overproducing TbPCNA or human PCNA (hPCNA) in the pathogenic protist Trypanosoma brucei triggers a dominant-negative phenotype of arrested proliferation. The mechanism for arresting T. brucei proliferation requires the overproduced PCNA orthologs to have functional intact SLiM-binding pocket. Sight-directed mutagenesis studies showed that T. brucei overproducing PCNA variants with disrupted SLiM-binding pockets grew normally. We hypothesized that chemically disrupting the SLiM-binding pocket would restore proliferation in T. brucei, overproducing PCNA orthologs. Testing this hypothesis is the proof-of-concept for a T. brucei-based PCNA screening assay. The assay design is to discover bioactive small molecules that restore proliferation in T. brucei strains that overproduce PCNA orthologs, likely by disrupting interactions in the SLiM-binding pocket. The pilot screen for this assay discovered two hit compounds that linked to predetermined PCNA targets. Compound #1, a known hPCNA inhibitor, had selective bioactivity to hPCNA overproduced in T. brucei, validating the assay. Compound #6 had promiscuous bioactivity for hPCNA and TbPCNA but is the first compound discovered with bioactivity for inhibiting TbPCNA.

Design of Class I/IV Bromodomain-Targeting Degraders for Chromatin Remodeling Complexes.

Targeted protein degradation is an emerging technology that can be used for modulating the activity of epigenetic protein targets. Among bromodomain-containing proteins, a number of degraders for the BET family have been developed, while non-BET bromodomains remain underexplored. Several of these proteins are subunits in chromatin remodeling complexes often associated with oncogenic roles. Here, we describe the design of class I (BPTF and CECR2) and IV (BRD9) bromodomain-targeting degraders based on two scaffolds derived from pyridazinone and pyrimidine-based heterocycles. We evaluate various exit vectors and linkers to identify analogues that demonstrate selectivity within these families. We further use an in-cell NanoBRET assay to demonstrate that these heterobifunctional molecules are cell-permeable, form ternary complexes, and can degrade nanoluciferase-bromodomain fusions. As a first example of a CECR2 degrader, we observe that our pyrimidine-based analogues degrade endogenous CECR2 while showing a smaller effect on BPTF levels. The pyridazinone-based compounds did not degrade BPTF when observed through Western blotting, further supporting a more challenging target for degradation and a goal for future optimization.

Phenyl Dihydrouracil: An Alternative Cereblon Binder for PROTAC Design.

Thalidomide and its analogues are frequently used in PROTAC design. However, they are known to be inherently unstable, undergoing hydrolysis even in commonly utilized cell culture media. We recently reported that phenyl glutarimide (PG)-based PROTACs displayed improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. Our optimization efforts, aiming to further improve the chemical stability and eliminate the racemization-prone chiral center in PG, led us to the development of phenyl dihydrouracil (PD)-based PROTACs. Here we describe the design and synthesis of LCK-directing PD-PROTACs and compare their physicochemical and pharmacological properties to those of the corresponding IMiD and PG analogues.

Preclinical evaluation of proteolytic targeting of LCK as a therapeutic approach in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and there is an unmet need for targeted therapies, especially for patients with relapsed disease. We have recently identified pre-T cell receptor and lymphocyte-specific protein tyrosine kinase (LCK) signaling as a common therapeutic vulnerability in T-ALL. LCK inhibitor dasatinib showed efficacy against T-ALL in preclinical studies and in patients with T-ALL; however, this is transient in most cases. Leveraging the proteolysis targeting chimera (PROTAC) approach, we developed a series of LCK degraders using dasatinib as an LCK ligand and phenyl-glutarimide as a cereblon-directing moiety. Our lead compound SJ11646 exhibited marked efficiency in cereblon-mediated LCK degradation in T-ALL cells. Relative to dasatinib, SJ11646 showed up to three orders of magnitude higher cytotoxicity in LCK-activated T-ALL cell lines and primary leukemia samples in vitro, with drastically prolonged suppression of LCK signaling. In vivo pharmacokinetic and pharmacodynamic profiling indicated a 630% increase in the duration of LCK suppression by SJ11646 over dasatinib in patient-derived xenograft models of T-ALL, which translated into its extended leukemia-free survival over dasatinib in vivo. Last, SJ11646 retained a high binding affinity to 51 human kinases, particularly ABL1, KIT, and DDR1, all of which are known drug targets in other cancers. Together, our dasatinib-based phenyl-glutarimide PROTACs are promising therapeutic agents in T-ALL and valuable tools for developing degradation-based therapeutics for other cancers.

Ca2+-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak.

The ability of mitochondria to buffer a rapid rise in cytosolic Ca2+ is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca2+ imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca2+ release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca2+. When the latter exceeded the organelle buffering capacity, an abrupt mitochondrial inner membrane permeabilization (MIMP) occurred, releasing matrix contents into the cytosol. MIMP was followed by cell death that was independent of Bcl-2 family members and inhibitable by the intracellular Ca2+ chelator BAPTA-AM. Cyclosporin A (CsA), capable of blocking the mitochondrial permeability transition (MPT), completely prevented cell death induced by m-3M3FBS. However, CsA acted upstream of mitochondria by preventing Ca2+ release from ER stores. Therefore, loss of Ca2+ intracellular balance and mitochondrial Ca2+ overload followed by MIMP induced a cell death process that is distinct from Bcl-2 family-regulated mitochondrial outer membrane permeabilization (MOMP). Further, the inhibition of cell death by CsA or its analogues can be independent of effects on the MPT.

Development of Potent and Selective Janus Kinase 2/3 Directing PG-PROTACs.

Aberrant activation of the JAK-STAT signaling pathway has been implicated in the pathogenesis of a range of hematological malignancies and autoimmune disorders. Here we describe the design, synthesis, and characterization of JAK2/3 PROTACs utilizing a phenyl glutarimide (PG) ligand as the cereblon (CRBN) recruiter. SJ10542 displayed high selectivity over GSPT1 and other members of the JAK family and potency in patient-derived ALL cells containing both JAK2 fusions and CRLF2 rearrangements.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) accounts for more than half of Philadelphia chromosome-like (Ph-like) ALL and is associated with a poor outcome in children and adults. Overexpression of CRLF2 results in activation of Janus kinase (JAK)-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of JAKs show variable and limited efficacy. Here, we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against JAKs. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of a series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers, and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity was tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded JAKs and potently killed CRLF2r cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1 and suppressed proliferation of CRLF2r ALL in vivo, e.g. compound 7 (SJ988497). Although dual JAK/GSPT1-degrading PROTACs were the most potent, the development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading, GSPT1-sparing PROTAC that demonstrated efficacy in the majority of kinase-driven xenografts that were otherwise unresponsive to type I JAK inhibitors, e.g. compound 8 (SJ1008030). Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL and highlight the interplay of JAK and GSPT1 degradation activity in this context.

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.