Casitas B-lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ligase that has an important role in effector T cell function, acting as a negative regulator of T cell, natural killer (NK) cell, and B cell activation. A discovery effort toward Cbl-b inhibitors was pursued in which a generative AI design engine, REINVENT, was combined with a medicinal chemistry structure-based design to discover novel inhibitors of Cbl-b. Key to the success of this effort was the evolution of the “Design” phase of the Design-Make-Test-Analyze cycle to involve iterative rounds of an in silico structure-based drug design, strongly guided by physics-based affinity prediction and machine learning DMPK predictive models, prior to selection for synthesis. This led to the accelerated discovery of a potent series of carbamate Cbl-b inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.

Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ligase that is responsible for repressing T-cell, natural killer (NK) cell, and B-cell activation. The robust antitumor activity observed in Cbl-b deficient mice arising from elevated T-cell and NK-cell activity justified our discovery effort toward Cbl-b inhibitors that might show therapeutic promise in immuno-oncology, where activation of the immune system can drive the recognition and killing of cancer cells. We undertook a high-throughput screening campaign followed by structure-enabled optimization to develop a novel benzodiazepine series of potent Cbl-b inhibitors. This series displayed nanomolar levels of biochemical potency, as well as potent T-cell activation. The functional activity of this class of Cbl-b inhibitors was further corroborated with ubiquitin-based cellular assays.

Aberrant activity of the histone methyltransferase polycomb repressive complex 2 (PRC2) has been linked to several cancers, with small-molecule inhibitors of the catalytic subunit of the PRC2 enhancer of zeste homologue 2 (EZH2) being recently approved for the treatment of epithelioid sarcoma (ES) and follicular lymphoma (FL). Compounds binding to the EED subunit of PRC2 have recently emerged as allosteric inhibitors of PRC2 methyltransferase activity. In contrast to orthosteric inhibitors that target EZH2, small molecules that bind to EED retain their efficacy in EZH2 inhibitor-resistant cell lines. In this paper we disclose the discovery of potent and orally bioavailable EED ligands with good solubilities. The solubility of the EED ligands was optimized through a variety of design tactics, with the resulting compounds exhibiting in vivo efficacy in EZH2-driven tumors.

Free Energy Perturbation (FEP) calculations can provide high-confidence predictions of the interaction strength between a ligand and its protein target. We sought to explore a series of triazolopyrimidines which bind to the EED subunit of the PRC2 complex as potential anticancer therapeutics, using FEP calculations to inform compound design. Combining FEP predictions with a late-stage functionalisation (LSF) inspired synthetic approach allowed us to rapidly evaluate structural modifications in a previously unexplored region of the EED binding site. This approach generated a series of novel triazolopyrimidine EED ligands with improved physicochemical properties and which inhibit PRC2 methyltransferase activity in a cancer-relevant G401 cell line.

The polycomb repressive complex 2(PRC2), consisting of the core subunits EED, EZH2, and SUZ12, is frequently overexpressed and deregulated in hematological and solid malignancies. Clinical validation for targeting the PRC2 complex has been demonstrated with several small molecule inhibitors of EED or EZH2 that are either approved or being evaluated in clinical trials in different cancer settings. Like other treatment modalities, single-agent activity is limited. Given that the PRC2 complex is involved in multiple diverse oncogenic/immune pathways, we thus explored the combination potential of inhibiting PRC2 in a wide array of biological contexts. To this end, we have developed a potent, selective, and orally bioavailable EED ligand. The compound binds to EED (K D =3.2 nM) and inhibits PRC2 enzyme activity (IC 50= 7nM). The compound inhibits proliferation of an EZH2 dependent Karpas422 GCB-DLBCL cell line (GI 50= 27nM) in vitro. In vivo, the inhibitor is well tolerated and drives tumor regression at 10 mpk and 50 mpk doses in a DLBCL xenograft model. Given the compound has favorable selectivity, potency and pharmacokinetic profile, we next evaluated the combinability of this EEDi with select compounds targeting diverse oncogenic pathways. We find that EED and PI3K/AKT inhibitors show marked combination activity in DLBCL, in part through downregulation of AKT signaling. Combination activity is also observed with the EED inhibitor in combination with the BTK inhibitor, acalabrutinib, in DLBCL, although this appears to be context dependent. In addition to DLBCL models, we have also assessed the therapeutic potential of EED inhibitor combinations in solid tumors. In SCLC, the EED inhibitor increased expression of SLFN11, a biomarker that has been linked to clinical response to a PARP inhibitor / temozolomide combination (Pietanza et al., 2018). Therefore, we evaluated the EED inhibitor in combination with the PARP inhibitor, olaparib, in an aggressive SCLC xenograft model and observed reduced tumor growth in the combination arm but not in monotherapy arms. In ovarian cancer, ARID1A mutations are proposed to sensitize to EZH2 and ATR inhibitors (Bitler et al., 2015, Williamson et al., 2016) which prompted evaluation of the EED inhibitor with the ATR inhibitor, AZD6738. Indeed, the combination resulted in tumor stasis whereas the monotherapies produced modest tumor growth inhibition. Last, we have evaluated the EED inhibitor alone, and in combination with a CTLA4 antibody, in a tumor immunity setting using an immunologically cold syngeneic melanoma model. Interestingly, we find that EEDi has significant anti-tumor activity alone, as compared with either control or CTLA4 antibody treatment alone. Together, these data show that while our EEDi has strong single-agent activity in vitro and in vivo, combining the EEDi together with inhibitors of select oncogenic pathways may bring deeper therapeutic response in a context dependent manner.

Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pK(D) similar to 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC(50) similar to 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI(50)] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.

Abstract Deregulation of the PRC2 components EZH2, SUZ12, and EED plays critical roles in driving aberrant hypermethylation of H3K27 and tumorigenicity of many solid and hematological malignancies. Although SAM competitive small molecule inhibitors of EZH2 show promising clinical activity in PRC2-dependent cancers, preclinical data suggests that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (Proteolysis Targeting Chimera) to efficiently target EED for elimination. Our EED-targeting PROTACs bind to EED (pKD= 9.02-9.27) and promote stable ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50= 8.11-8.17) that results in a decrease in H3K27me3 levels in cells. Interestingly, EED-targeting PROTACs induce rapid degradation of EED, as well as its associated proteins, including EZH2 and SUZ12 in the PRC2 complex. Inhibition of the ubiquitin proteasome pathway abrogates PROTAC-mediated degradation of EED and its associated proteins. Furthermore, the EED targeting PROTACs selectively inhibit proliferation and survival of PRC2-dependent cancer cells (GI50= 49-58 nM). In summary, our data demonstrate a novel therapeutic modality in treating PRC2 dependent cancer through PROTAC mediated platform. Citation Format: Jessie Hao-Ru Hsu, Timothy Rasmusson, James Robinson, Fiona Pachl, Jon Read, Sameer Kawatkar, Daniel H O'Donovan, Sharan Bagal, Erin Code, Philip Rawlins, Argyrides Argyrou, Ronald Tomlinson, Ning Gao, Xiahui Zhu, Elisabetta Chiarparin, Kelly Jacques, Minhui Shen, Haley Woods, Emma Bednarski, David M. Wilson, Lisa Drew, M. Paola Castaldi, Stephen Fawell, Andrew Bloecher. EED targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A09. doi:10.1158/1535-7163.TARG-19-LB-A09

Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates chromatin state and gene expression by methylating histone H3 lysine 27. EZH2 is overexpressed or mutated in various hematological malignancies and solid cancers. Our previous efforts to identify inhibitors of PRC2 methyltransferase activity by high-throughput screening (HTS) resulted in large numbers of false positives and thus a significant hit deconvolution challenge. More recently, others have reported compounds that bind to another PRC2 core subunit, EED, and allosterically inhibit EZH2 activity. This mechanism is particularly appealing as it appears to retain potency in cell lines that have acquired resistance to orthosteric EZH2 inhibition. By designing a fluorescence polarization probe based on the reported EED binding compounds, we were able to quickly and cleanly re-triage our previously challenging HTS hit list and identify novel allosteric PRC2 inhibitors.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

Inhibition of CDK9, an enzyme that regulates transcriptional elongation, can lead to apoptosis in cancer cells. Thus CDK9 inhibitors are potential new anti‐cancer agents. In order to more fully characterize novel CDK9 inhibitors that we are developing we wanted to be able to perform kinetic analyses including the determination of kinetic on and off rates. The CDK9/CyclinT kinase domain exhibited rapid loss of ligand binding ability when immobilized on a SPR chip, preventing collection of kinetic data. To improve stability, a series of amino acid changes were made based on the crystal structure. These included surface residue changes of both the CDK9 kinase domain and Cyclin T, and combinations of the various changes (up to 7 changes in CDK9 and 5 in Cyclin T). Modified genes were synthesized, a matrix of modified CDK9 and cyclin T combinations defined (18 conditions) and expressed in insect cells. Constructs were evaluated by expression levels, purification yield, ability to concentrate protein to high levels, increased melting temperature, conjugation and stability during SPR. The original CDK9/Cyclin T complex lost activity minutes after conjugation to the SPR chip whereas the “thermostabilized” CDK9 (7 residue modified) Cyclin T (5 residue modified) complex is active for 2.5 days after immobilization allowing for determination of on and off rates of CDK9 Cyclin T inhibitors.

Significance The identification of negamycin’s binding site within helix 34 of the small subunit head domain and the elucidation of its mechanism of action during messenger RNA decoding provide a physical framework for exploring structure–activity relationships of this largely unexplored antibiotic class. These findings lay the foundation for the rational design of improved negamycin analogs that may one day serve as potent antibacterial agents in the clinic.

Abstract The PIM serine/threonine kinase family, composed of three highly homologous members, PIM-1, PIM-2 and PIM-3, are upregulated in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Over-expression of PIM-1 or PIM-3 has also been observed several solid tumors, in particular prostate, pancreatic, gastric, bladder and hepatocellular cancers. PIM kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways, thereby mediating cell proliferation and survival. We have identified novel, potent and highly selective inhibitors of the PIM family kinases. The synthesis, X-ray crystallographic binding mode, and SAR of this benzylidene-1,3-thiazolidine-2,4-dione series are described. Examples from this series exhibit single digit nanomolar potency against all three PIMs, and have been shown to be selective across a panel of more than 440 kinases, with inhibition found for only approximately 3% of the panel, and at least 10-fold selectivity over kinases outside the PIM family. The compounds additionally are stable in rat microsomes, have high aqueous solubilities, and are not potent against the hERG ion channel. The series has antiproliferative activity in a panel of AML cells, has excellent pre-clinical pharmacokinetic properties, and lead compounds have shown strong tumor growth inhibition in vivo in mouse AML xenograft models. From this series, AZD1208 has recently entered Phase I clinical trials for AML and solid tumors. Citation Format: Michelle L. Lamb, Les A. Dakin, Michael H. Block, Huawei Chen, Erin Code, James E. Dowling, Xiaomei Feng, Andrew D. Ferguson, Isabelle Green, Alexander W. Hird, Tina Howard, Dennis Huszar, Erika K. Keeton, Paul D. Lyne, Hannah Pollard, Michael Rooney, Jon Read, Allan J. Wu, Tao Zhang, Xiaolan Zheng. Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2013-2353

Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a Ki of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics.

Abstract KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling, a strategy employed by cancer cells to avoid multipolar mitosis in the presence of amplified centrosomes. However, its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. We have recently reported the first small molecule inhibitor of KIFC1, AZ82 [1]. AZ82 binds specifically to the KIFC1/microtubule (MT) binary complex, and inhibits the MT-stimulated KIFC1 enzymatic activity with a KI of 0.043 µM. AZ82 effectively engaged with the minus-end directed KIFC1 motor in HeLa cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5 by AZD4877, consistent with what was observed with genetic knock down of KiFC1 by siRNA. Additionally, treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Here we further describe the chemistry approach and related structure-activity relationships that led to the discovery of AZ82. [1] http://pubs.acs.org/doi/abs/10.1021/cb400186w Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C55. Citation Format: Michelle L. Lamb, Jiaquan Wu, Keith Mikule, Wendy Wang, Nancy Su, Philip Petteruti, Farzin Gharahdaghi, Erin Code, Xiahui Zhu, Kelly Jacques, Zhongwu Lai, Tao Zhang, David Boulay, Gurmit Grewal, Nicholas Keen, Bin Yang, Claudio Chuaqui, Claudio Chuaqui, Huawei Chen. Discovery and optimization of inhibitors of the KIFC1 motor protein. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C55.

Abstract Despite the success of Crizotinib in treating NSCLC patients with EML4-ALK fusions, cancers eventually develop resistance via a variety of mechanisms including mutations in the ATP binding site of the kinase domain. A series of 5-aminopyrazol-imidazopyridine compounds were identified to potently inhibit anaplastic lymphoma kinase and were found to be active against a number of mutations in vitro but suffered from strong Cyp inhibition and were found to be metabolically unstable by incubation in human hepatocytes and microsomes. Installation of a pyrimary alcohol group on the heteroaryl ethyl group of the scaffold consistently improved Cyp3A4 liability, and led to improvement in physical and DMPK properties as well as improvement of their metabolic stability in human Heps. The lead compound from this series was orally administrated to SCID mice in a Del xenograft model and achieved greater than 90% phospho-ALK inhibition over 6 hours post dose at 10 mg/kg dose. The lead compound was subsequently tested against Crizotinib-resistant ALK mutations in enzyme assays, and was shown to inhibit the clinically relevant ALK mutations, including L1196M. In enzyme assays, it was inactive against G1269S mutation, consistent with modeling studies. Subsequent profiling in an FDCP cell line over expressing ALK G1269S mutation showed potent anti-proliferative activity, suggesting off-target activity. Further characterization of this series identified AurB inhibition to be a key cell cycle kinase responsible for the off-target activity in the engineered cell line. The off target activity hinders it from assessing the primary pharmacology responsible for its preclinical efficacy in disease relevant models. In conclusion, we have identified a novel orally bioavailable compound that is a potent dual active ALK and AurB inhibitor that is active against clinically-relevant gatekeeper mutant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3910. doi:1538-7445.AM2012-3910

Novel substituted benzylidene-1,3-thiazolidine-2,4-diones (TZDs) have been identified as potent and highly selective inhibitors of the PIM kinases. The synthesis and SAR of these compounds are described, along with X-ray crystallographic, anti-proliferative, and selectivity data.

SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.

Structure activity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.