Discovery, Optimization, and Biological Evaluation of Arylpyridones as 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.

Discovery of a Novel Benzodiazepine Series of Cbl-b Inhibitors for the Enhancement of Antitumor Immunity.

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.

Diverse, Potent, and Efficacious Inhibitors That Target the EED Subunit of the Polycomb Repressive Complex 2 Methyltransferase.

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 in the design of EED ligands as inhibitors of polycomb repressive complex 2 (PRC2) methyltransferase.

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.

EED-Targeted PROTACs Degrade EED, EZH2, and SUZ12 in the PRC2 Complex.

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 (pKD ∼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 ∼ 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 [GI50] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.

Rapid Identification of Novel Allosteric PRC2 Inhibitors.

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.

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6.

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.

Discovery of Pyrazolo[1,5-a]pyrimidine B-Cell Lymphoma 6 (BCL6) Binders and Optimization to High Affinity Macrocyclic Inhibitors.

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.

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.

Discovery and mechanistic study of a small molecule inhibitor for motor protein KIFC1.

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.

Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases.

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.

Discovery of (+)-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), a kinesin spindle protein inhibitor and potential anticancer agent.

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.

Biochemical characterization of human SET and MYND domain-containing protein 2 methyltransferase.

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.