Synthesis of Pyrazole-Based Macrocycles Leads to a Highly Selective Inhibitor for MST3.

MST1, MST2, MST3, MST4, and YSK1 are conserved members of the mammalian sterile 20-like serine/threonine (MST) family that regulate cellular functions such as proliferation and migration. The MST3 isozyme plays a role in regulating cell growth and apoptosis, and its dysregulation has been linked to high-grade tumors. To date, there are no isoform-selective inhibitors that could be used for validating the role of MST3 in tumorigenesis. We designed a series of 3-aminopyrazole-based macrocycles based on the structure of a promiscuous inhibitor. By varying the moieties targeting the solvent-exposed region and optimizing the linker, macrocycle JA310 (21c) was synthesized. JA310 exhibited high cellular potency for MST3 (EC50 = 106 nM) and excellent kinome-wide selectivity. The crystal structure of the MST3-JA310 complex provided intriguing insights into the binding mode, which is associated with large-scale structural rearrangements. In summary, JA310 demonstrates the utility of macrocyclization for the design of highly selective inhibitors and presents the first chemical probe for MST3.

Discovery of 3-Amino-1H-pyrazole-Based Kinase Inhibitors to Illuminate the Understudied PCTAIRE Family.

The PCTAIRE subfamily belongs to the CDK (cyclin-dependent kinase) family and represents an understudied class of kinases of the dark kinome. They exhibit a highly conserved binding pocket and are activated by cyclin Y binding. CDK16 is targeted to the plasma membrane after binding to N-myristoylated cyclin Y and is highly expressed in post-mitotic tissues, such as the brain and testis. Dysregulation is associated with several diseases, including breast, prostate, and cervical cancer. Here, we used the N-(1H-pyrazol-3-yl)pyrimidin-4-amine moiety from the promiscuous inhibitor 1 to target CDK16, by varying different residues. Further optimization steps led to 43d, which exhibited high cellular potency for CDK16 (EC50 = 33 nM) and the other members of the PCTAIRE and PFTAIRE family with 20-120 nM and 50-180 nM, respectively. A DSF screen against a representative panel of approximately 100 kinases exhibited a selective inhibition over the other kinases. In a viability assessment, 43d decreased the cell count in a dose-dependent manner. A FUCCI cell cycle assay revealed a G2/M phase cell cycle arrest at all tested concentrations for 43d, caused by inhibition of CDK16.

Illuminating the Dark: Highly Selective Inhibition of Serine/Threonine Kinase 17A with Pyrazolo[1,5-a]pyrimidine-Based Macrocycles.

Serine/threonine kinase 17A (death-associated protein kinase-related apoptosis-inducing protein kinase 1─DRAK1) is a part of the death-associated protein kinase (DAPK) family and belongs to the so-called dark kinome. Thus, the current state of knowledge of the cellular function of DRAK1 and its involvement in pathophysiological processes is very limited. Recently, DRAK1 has been implicated in tumorigenesis of glioblastoma multiforme (GBM) and other cancers, but no selective inhibitors of DRAK1 are available yet. To this end, we optimized a pyrazolo[1,5-a]pyrimidine-based macrocyclic scaffold. Structure-guided optimization of this macrocyclic scaffold led to the development of CK156 (34), which displayed high in vitro potency (KD = 21 nM) and selectivity in kinomewide screens. Crystal structures demonstrated that CK156 (34) acts as a type I inhibitor. However, contrary to studies using genetic knockdown of DRAK1, we have seen the inhibition of cell growth of glioma cells in 2D and 3D culture only at low micromolar concentrations.

Synthetic Opportunities and Challenges for Macrocyclic Kinase Inhibitors.

Macrocycles are typically cyclic variants of inhibitors derived from uncyclized canonical molecules or from natural products. For medicinal chemistry, drug-like macrocycles have received increasing interest over the past few years, since it has been demonstrated that macrocyclization can favorably alter the biological and physiochemical properties as well as selectivity in comparison to the acyclic analogue. Recent drug approvals such as Lorlatinib, glecaprevir, or voxilaprevir underline the clinical relevance of drug-like macrocycles. However, the synthesis of drug-like macrocycles can be challenging, since the ring-closing reaction is generally challenging with yields depending on the size and geometry of the bridging linker. Nevertheless, macrocycles are one opportunity to expand the synthetic toolbox for medicinal chemistry to provide bioactive molecules. Therefore, we reviewed the past literature of drug-like macrocycles highlighting reactions that have been successfully used for the macrocyclization. We classified the cyclization reactions by their type, ring-size, yield, and macrocyclization efficiency index.