Synthesis of Novel Aporphine-Inspired Neuroreceptor Ligands

Aporphines are a group of tetracyclic alkaloids that belong to the ubiquitous tetrahydroisoquinoline family. The aporphine template is known to be associated with a range of biological activities. Aporphines have been explored as antioxidants, anti-tuberculosis, antimicrobial and anticancer agents. Within the Central Nervous Systems (CNS), aporphine alkaloids are known to possess high affinity for several clinically valuable targets including dopamine receptors (predominantly D1 and D2), serotonin receptors (5-HT1A and 5-HT7) and α adrenergic receptors. Aporphines are also inhibitors of the acetylcholinesterase enzyme – a clinical target for the treatment of Alzheimer’s disease. Considering the diverse profile of aporphine alkaloids at CNS receptors they can be considered as “privileged scaffold” for the design of CNS drugs. The aporphine alkaloid nantenine is a 5-HT2A receptor antagonist and has moderate affinity for the 5-HT2A receptor. Selective 5-HT2A antagonists have therapeutic potential for the treatment of a number of neuropsychiatric disorders including depression, schizophrenia and sleep disorders. The aporphine core of nantenine serves as a valuable lead for the identification of selective 5-HT2A antagonists. In order to understand the structural tolerance of the aporphine core required for 5-HT2A antagonism an exhaustive Structure Activity Relationship (SAR) study was designed.Accordingly, a diverse library of nantenine analogues was synthesized and evaluated for affinity at the 5-HT2A receptor. Results from the SAR studies demonstrate that the nitrogen atom of nantenine is required for affinity and that introduction of a phenyl ring at the C4 position is detrimental for 5-HT2A receptor affinity. At the C3 position, introduction of halogen atoms is beneficial for 5-HT2A antagonistic activity. Furthermore, a library of C3 analogues having hydrophobic substituents as well as ring D indole analogues is currently being evaluated for affinity at the 5-HT2A receptors. These compounds will further expand our understanding of the tolerance of the aporphine core required for 5-HT2A antagonism. In order to rationalize the affinity of certain high affinity ligands, molecular docking studies were conducted. Selected compounds were docked into a homology model of the 5-HT2A receptor to extract information about possible binding modes. Based on results of these studies, it is concluded that the interaction of C3 halogenated aporphine analogues with Phe339/Phe340 residues might be responsible for their enhanced affinity. Information obtained from molecular docking studies is being utilized for design of advanced generations of analogues. Finally, a novel series of flexible tris-(phenylalkyl)amines were synthesized and evaluated to test the importance of a rigid aporphine core as well as incorporation of N-phenylalkyl substituents. These compounds featuring a halogen substituent in ring C, were found to have high affinity and selectivity for the 5-HT2B receptor, with some of the compounds being more potent than the selective 5-HT2B antagonist SB200646. Results from this study indicate that ring C of these compounds is generally tolerant for halogen substitution. The synthetic feasibility of this newly identified template ( 4 high-yielding synthetic steps from commercially available materials) makes this scaffold attractive for the synthesis of larger libraries of analogs and promise for optimization of 5-HT2B affinity and selectivity

WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of LRP6 and establish a negative feedback loop

beta-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity2617993FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/50724-5; 2016/17469-0M.B.M. acknowledges support from the NIH (RO1-CA187799 and U24-DK116204-01). M.J.A. received financial support from NIH T32 Predoctoral Training Grants in Pharmacology (T32-GM007040-43 and T32-GM007040-42), an Initiative for Maximizing Student Diversity Grant (R25-GM055336-16), and the NIH National Cancer Institute (NCI) NRSA Predoctoral Fellowship to Promote Diversity in Health-Related Research (F31CA228289). M.P.W. received support from the Lymphoma Research Foundation (337444) and the NIH (T32-CA009156-35). Y.N. was supported by grants-in-aid from the Japan Society for the Promotion of Science (JSPS) (15KK0356 and 16K11493). T.T. was supported by the Howard Hughes Medical Institute Gilliam Fellowship for Advanced Study. M.V.G. was supported by Cancer Research UK (grants C7379/A15291 and C7379/A24639 to Mariann Bienz). The UNC Flow Cytometry Core Facility is supported in part by Cancer Center Core Support Grant P30 CA016086 to the UNC Lineberger Comprehensive Cancer Center, and research reported in this publication was supported by the Center for AIDS Research (award number 5P30AI050410), and the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The Structural Genomics Consortium (SGC) is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, the Eshelman Institute for Innovation, Genome Canada, the Innovative Medicines Initiative (European Union [EU]/European Federation of Pharmaceutical Industries and Associations [EFPIA]) (ULTRA-DD grant no. 115766), Janssen, Merck & Company, Merck KGaA, Novartis Pharma AG, the Ontario Ministry of Economic Development and Innovation, Pfizer, the São Paulo Research Foundation (FAPESP) (2013/50724-5), Takeda, and the Wellcome Trust (106169/ZZ14/Z). R.R.R. received financial support from FAPESP (2016/17469-0). We would also like to thank Claire Strain-Damerell and Pavel Savitsky for cloning various mutants of AAK1 and BMP2K proteins that were used in the crystallization trials. Additionally, we thank Dr. Sean Conner for providing the AAK1 plasmids, Dr. Stephane Angers for kindly providing the HEK293T DVL TKO cells, and Dr. Mariann Bienz for providing comments and feedback. We would like to thank members of the Major laboratory for their feedback and expertise regarding experimental design and project directio

A Chemical Probe Targeting AAK1 and BMP2K

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization of this 3-acylaminoindazole scaffold furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.</p