Discovery of 9H-pyrimido[4,5-b]indole derivatives as dual RET/TRKA inhibitors.

Aberrant RET kinase signaling is activated in numerous cancers including lung, thyroid, breast, pancreatic, and prostate. Recent approvals of selective RET inhibitors, pralsetinib and selpercatinib, has shifted the focus of RET kinase drug discovery programs towards the development of selective inhibitors. However, selective inhibitors invariably lose efficacy as the selective nature of the inhibitor places Darwinian-like pressure on the tumor to bypass treatment through the selection of novel oncogenic drivers. Further, selective inhibitors are restricted for use in tumors with specific genetic backgrounds that do not encompass diverse patient classes. Here we report the identification of a pyrimido indole RET inhibitor found to also have activity against TRK. This selective dual RET/TRK inhibitor can be utilized in tumors with both RET and TRK genetic backgrounds and can also provide blockade of NTRK-fusions that are selected for from RET inhibitor treatments. Efforts towards developing dual RET/TRK inhibitors can be beneficial in terms of encompassing more diverse patient classes while also achieving blockade against emerging resistance mechanisms.

Kinase inhibitor macrocycles: a perspective on limiting conformational flexibility when targeting the kinome with small molecules.

Methods utilized for drug discovery and development within the kinome have rapidly evolved since the approval of imatinib, the first small molecule kinase inhibitor. Macrocycles have received increasing interest as a technique to improve kinase inhibitor drug properties evident by the FDA approvals of lorlatinib, pacritinib, and repotrectinib. Compared to their acyclic counterparts, macrocycles can possess improved pharmacodynamic and pharmacokinetic properties. This review highlights clinical success stories when implementing macrocycles in kinase-based drug discovery and showcases that macrocyclization is a clinically validated drug discovery strategy when targeting the kinome.

Discovery of the DNA-PKcs inhibitor DA-143 which exhibits enhanced solubility relative to NU7441.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a vital role in DNA damage repair and lymphocyte function, presenting a significant target in cancer and immune diseases. Current DNA-PKcs inhibitors are undergoing Phase I/II trials as adjuncts to radiotherapy and chemotherapy in cancer. Nevertheless, clinical utility is limited by suboptimal bioavailability. This study introduces DNA-PKcs inhibitors designed to enhance bioavailability. We demonstrate that a novel DNA-PKcs inhibitor, DA-143, surpasses NU7441 in aqueous solubility as well as other available inhibitors. In addition, DA-143 displayed an improvement in DNA-PKcs inhibition relative to NU7441 achieving an IC50 of 2.5 nM. Consistent with current inhibitors, inhibition of DNA-PKcs by DA-143 resulted in increased tumor cell sensitivity to DNA-damage from chemotherapy and inhibition of human T cell function. The improved solubility of DA-143 is critical for enhanced efficacy at reduced doses and facilitates more effective evaluation of DNA-PKcs inhibition in both preclinical and clinical development.

Bifunctional Inhibitor Reveals NEK2 as a Therapeutic Target and Regulator of Oncogenic Pathways in Lymphoma.

Expression of the serine/threonine kinase never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is essential for entry into mitosis via its role in facilitating centrosome separation. Its overactivity can lead to tumorigenesis and drug resistance through the activation of several oncogenic pathways, including AKT. Although the cancer-enabling activities of NEK2 are documented in many malignancies, including correlations with poor survival in myeloma, breast, and non-small cell lung cancer, little is known about the role of NEK2 in lymphoma. Here, in tumors from patients with diffuse large B-cell lymphoma (DLBCL), the most common, aggressive non-Hodgkin lymphoma, we found a high abundance of NEK2 mRNA and protein associated with an inferior overall survival. Using our recently developed NEK2 inhibitor, NBI-961, we discovered that DLBCL cell lines and patient-derived cells exhibit a dependency on NEK2 for their viability. This compromised cell fitness was directly attributable to efficient NEK2 inhibition and proteasomal degradation by NBI-961. In a subset of particularly sensitive DLBCL cells, NBI-961 induced G2/mitosis arrest and apoptosis. In contrast, an existing indirect NEK2 inhibitor, INH154, did not prevent NEK2 autophosphorylation, induce NEK2 proteasomal degradation, or affect cell viability. Global proteomics and phospho-proteomics revealed that NEK2 orchestrates cell-cycle and apoptotic pathways through regulation of both known and new signaling molecules. We show the loss of NEK2-sensitized DLBCL to the chemotherapy agents, doxorubicin and vincristine, and effectively suppressed tumor growth in mice. These studies establish the oncogenic activity of NEK2 in DLBCL and set the foundation for development of anti-NEK2 therapeutic strategies in this frequently refractory and relapse-prone cancer.

An updated patent review of rearranged during transfection (RET) kinase inhibitors (2016-present).

INTRODUCTION: Rearranged during transfection (RET) is a transmembrane receptor tyrosine kinase. Aberrations in RET signaling due to mutations, gene fusions, or overexpression can lead to carcinomas. Six inhibitors have been approved for the treatment of RET-driven cancers: vandetanib, cabozantinib, lenvatinib, sorafenib, selpercatinib, and pralsetinib. Only selpercatinib and pralsetinib have been developed specifically for RET, while the remaining are multikinase inhibitors. Several other RET targeted candidates are under clinical development. AREAS COVERED: This review covers recent patent literature describing small molecules that are active against RET since 2016 till present. EXPERT OPINION: RET represents a major therapeutic target as its alterations occur in nearly 2% of all cancers. Recent approvals for RET targeted therapy have been developed specifically to target the RET oncogene. These approvals represent a paradigm shift from the last decade to now focus on the development of selective RET inhibitors rather than multikinase inhibitors. These newly approved RET inhibitors still have clinical issues with drug resistance. It is imperative that the next iteration of RET inhibitors are developed to block common treatment-resistant mutations. To accomplish this, RET inhibitors should be developed in concert with genomic profiling to ensure the most relevant clinical mutations are targeted.

FLT3 inhibitors for acute myeloid leukemia: successes, defeats, and emerging paradigms.

FLT3 mutations are one of the most common genetic aberrations found in nearly 30% of acute myeloid leukemias (AML). The mutations are associated with poor prognosis despite advances in the understanding of the biological mechanisms of AML. Numerous small molecule FLT3 inhibitors have been developed in an effort to combat AML. Even with the development of these inhibitors, the five-year overall survival for newly diagnosed AML is less than 30%. In 2017, midostaurin received FDA approval to treat AML, which was the first approved FLT3 inhibitor in the U.S. and Europe. Following, gilteritinib received FDA approval in 2018 and in 2019 quizartinib received approval in Japan. This review parallels these clinical success stories along with other pre-clinical and clinical investigations of FLT3 inhibitors.

Targeting a Novel G-Quadruplex in the CARD11 Oncogene Promoter with Naptho(2,1-b)furan-1-ethanol,2-nitro- Requires the Nitro Group.

The aggressive nature of the activated B cell such as (ABC) subtype of diffuse large B cell (DLBCL) is frequently associated with altered B cell Receptor (BCR) signaling through the activation of key components including the scaffolding protein, CARD11. Most inhibitors, such as ibrutinib, target downstream BCR kinases with often modest and temporary responses for DLBCL patients. Here, we pursue an alternative strategy to target the BCR pathway by leveraging a novel DNA secondary structure to repress transcription. We discovered that a highly guanine (G)-rich element within the CARD11 promoter forms a stable G-quadruplex (G4) using circular dichroism and polymerase stop biophysical techniques. We then identified a small molecule, naptho(2,1-b)furan-1-ethanol,2-nitro- (NSC373981), from a fluorescence-resonance energy transfer-based screen that stabilized CARD11 G4 and inhibited CARD11 transcription in DLBCL cells. In generating and testing analogs of NSC373981, we determined that the nitro group is likely essential for the downregulation of CARD11 and interaction with CARD11 G4, and the removal of the ethanol side chain enhanced this activity. Of note, the expression of BCL2 and MYC, two other key oncogenes in DLBCL pathology with known promoter G4 structures, were often concurrently repressed with NSC373981 and the highly potent R158 analog. Our findings highlight a novel approach to treat aggressive DLBCL by silencing CARD11 gene expression that warrants further investigation.

Targeting Rearranged during Transfection in Cancer: A Perspective on Small-Molecule Inhibitors and Their Clinical Development.

Rearranged during transfection (RET) is a receptor tyrosine kinase essential for the normal development and maturation of a diverse range of tissues. Aberrant RET signaling in cancers, due to RET mutations, gene fusions, and overexpression, results in the activation of downstream pathways promoting survival, growth, and metastasis. Pharmacological manipulation of RET is effective in treating RET-driven cancers, and efforts toward developing RET-specific therapies have increased over the last 5 years. In 2020, RET-selective inhibitors pralsetinib and selpercatinib achieved clinical approval, which marked the first approvals for kinase inhibitors specifically developed to target the RET oncoprotein. This Perspective discusses current development and clinical applications for RET precision medicine by providing an overview of the incremental improvement of kinase inhibitors for use in RET-driven malignancies.

A Combined Infrared Ion Spectroscopy and Computational Chemistry Study of Hydroxyproline Isomers.

Hydroxyproline is a common variation of proline, with diverse biological roles. The hydroxylation of proline gives rise to several (natural and/or synthetic) isomeric forms, including both positional isomers and stereoisomers. While mass spectrometry is widely touted as a very selective analytical technique, the identification of closely related isomers often poses a challenge. In these cases, allied technologies become helpful in providing full characterization. Here, infrared multiple photon dissociation (IRMPD) spectroscopy is used to differentiate between three isomers, namely cis-3-hydroxyproline, cis-4-hydroxyproline, and trans-4-hydroxyproline. In contrast to the protonated species which show only minor variations in their IRMPD spectra, lithiated species were found to display significant spectral differences, making their differentiation more straightforward. The conformational origin of these spectral differences was investigated by complementary quantum-chemical calculations.