X-ray Crystal Structure-Guided Design and Optimization of 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile Scaffold as a Potent and Orally Active Monopolar Spindle 1 Inhibitor.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile scaffold. Our lead optimization was guided by key X-ray crystal structure analysis. In vivo evaluation of candidate (9) is shown to effectively mitigate human TNBC cell proliferation.

Paralog Specificity Determines Subcellular Distribution, Action Mechanism, and Anticancer Activity of TRAP1 Inhibitors.

Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.

Suppression of the metastatic spread of breast cancer by DN10764 (AZD7762)-mediated inhibition of AXL signaling.

Breast cancer is the most common malignant disease occurring in women and represents a substantial proportion of the global cancer burden. In these patients, metastasis but not the primary tumor is the main cause of breast cancer-related deaths. Here, we report the novel finding that DN10764 (AZD7762, a selective inhibitor of checkpoint kinases 1 and 2) can suppress breast cancer metastasis. In breast cancer cells, DN10764 inhibited cell proliferation and GAS6-mediated AXL signaling, consequently resulting in suppressed migration and invasion. In addition, DN10764 induced caspase 3/7-mediated apoptosis in breast cancer cells and inhibited tube formation of human umbilical vein endothelial cells. Finally, DN10764 significantly suppressed the tumor growth and metastasis of breast cancer cells in in vivo metastasis models. Taken together, these data suggest that therapeutic strategies targeting AXL in combination with systemic therapies could improve responses to anti-cancer therapies and reduce breast cancer recurrence and metastases.

Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL.

Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c-ABL and BCR-ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.

Catalytic in vivo protein knockdown by small-molecule PROTACs.

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Combinatorial assembly of small molecules into bivalent antagonists of TrkC or TrkA receptors.

A library of peptidomimetics was assembled combinatorially into dimers on a triazine-based core. The pharmacophore corresponds to ß-turns of the neurotrophin polypeptides neurotrophin-3 (NT-3), nerve growth factor (NGF), or brain-derived neurotrophic factor (BDNF). These are the natural ligands for TrkC, TrkA, and TrkB receptors, respectively. The linker length and the side-chain orientation of each monomer within the bivalent mimics were systematically altered, and the impact of these changes on the function of each ligand was evaluated. While the monovalent peptidomimetics had no detectable binding or bioactivity, four bivalent peptidomimetics (2c, 2d, 2e, 3f) are selective TrkC ligands with antagonistic activity, and two bivalent peptidomimetics (1a, 1b) are TrkC and TrkA ligands with antagonistic activity. All these bivalent compounds block ligand-dependent receptor activation and cell survival, without affecting neuritogenic differentiation. This work adds to our understanding of how the neurotrophins function through Trk receptors, and demonstrates that peptidomimetics can be designed to selectively disturb specific biological signals, and may be used as pharmacological probes or as therapeutic leads. The concept of altering side-chain, linker length, and sequence orientation of a subunit within a pharmacophore provides an easy modular approach to generate larger libraries with diversified bioactivity.

Small Molecule Ligands For Active Targeting Of TrkC-expressing Tumor Cells.

A small molecule motif was used in "active targeting" to deliver cytotoxic substances into tumor cells that express the TrkC receptor. Underlying this study was the hypothesis that internalization of targeted conjugates into cells would be facile if mediated by receptor binding and receptor-ligand internalization. Initial experiments using 6-mercaptopurine gave encouraging data, but demonstrated the importance of maintaining solubility and high cytotoxicity. Conjugates of the targeting agent with a cytotoxic rosamine (similar to a rhodamine) were more successful. Targeting of TrkC was observed, validated in a series of competition experiments featuring other TrkC ligands, and accumulation into lysosomes was observed, as expected for receptor-mediated internalization.

Minimalist and universal peptidomimetics.

Many "new generation" peptidomimetics are designed to present amino acid side chains only; they do not have structural features that resemble peptide main chains. These types of molecules have frequently been presented in the literature as mimics of specific secondary structures. However, many "side-chain only" peptidomimetics do not rest in single conformational states, but exist in a limited number of freely interconverting forms. These different conformations may resemble different secondary structures, so referring to them as, for instance, turn- or helical-mimics understates the ways they could adapt to various binding situations. Sets of scaffolds that can be used to mimic aspects of nearly every secondary structure, i.e. universal peptidomimetics, can be constructed. These may assume a privileged place in library design, particularly in high throughput screening for pharmacological probes for which binding conformations, or even the target itself, is unknown at the time the library is designed (critical review, 101 references).

In glaucoma the upregulated truncated TrkC.T1 receptor isoform in glia causes increased TNF-alpha production, leading to retinal ganglion cell death.

PURPOSE: Glaucoma is a distinct neuropathy characterized by the chronic and progressive death of retinal ganglion cells (RGCs). The etiology of RGC death remains unknown. Risk factors for glaucomatous RGC death are elevated intraocular pressure and glial production of tumor necrosis factor-alpha (TNF-α). Previously, the authors showed that glaucoma causes a rapid upregulation of a neurotrophin receptor truncated isoform lacking the kinase domain, TrkC.T1, in retina. Here they examined the biological role of TrkC.T1 during glaucoma progression. METHODS: Rat and mouse models of chronic ocular hypertension were used. Immunofluorescence Western blot analysis and in situ mRNA hybridization were used to identify cells upregulating TrkC.T1. A genetic model of engineered mice lacking TrkC.T1 (TrkC.T1(-/-)) was used to validate a role for this receptor in glaucoma. Pharmacologic studies were conducted to evaluate intravitreal delivery of agonists or antagonists of TrkC.T1, compared with controls, during glaucoma. Surviving RGCs were quantified by retrograde-labeling techniques. Production of neurotoxic TNF-α and α2 macroglobulin were quantified. RESULTS: TrkC.T1 was upregulated in retinal glia, with a pattern similar to that of TNF-α. TrkC.T1(-/-) mice had normal retinas. However, during experimental glaucoma, TrkC.T1(-/-) mice had lower rates of RGC death and produced less TNF-α than wild-type littermates. In rats with glaucoma, the pharmacologic use of TrkC antagonists delayed RGC death and reduced the production of retinal TNF-α. CONCLUSIONS: TrkC.T1 is implicated in glaucomatous RGC death through the control of glial TNF-α production. Overall, the data point to a paracrine mechanism whereby elevated intraocular pressure upregulated glial TrkC.T1 expression in glia; TrkC.T1 controlled glial TNF-α production, and TNF-α caused RGC death.