Design,Synthesis,and Biological Evaluation Of Small-molecule PROTACs For α_1A-Adrenergic Receptor

Proteolysis Targeting Chimeras(PROTACs)technology is an emerging drug discovery paradigm.Small-molecule PROTACs contain two ligands that are connected via a linker.One ligand would bind with the target protein,while the other one would recruit E3 ubiquitin ligase.Therefore,small-molecule PROTACs are selective bifunctional molecules that recruit an E3 ubiquitin ligase to direct the target protein into the Ubiquitin-proteasome system(UPS),thereby selectively reducing the level of the target protein by the 26S proteasome degradation.Protein knockdown by small-molecule PROTACs would provide a new modality to target a variety of proteins for degradation in cells,which could be applied to novel drug discovery and development.Up to now,small-molecule PROTACs have made remarkable advances since they have been used for inducing the degradation of many pathogenic proteins,including AR,RIPK2,ERRα,RAR,BRD2/3/4,TACC3,etc.Compared with the traditional inhibitor-based approach,small-molecule PROTACs have many advantages:(1)since ligands only need to bind the target protein and don’t need to block a functional site,it opened up new possibilities for those so-called"undruggable" proteins,albeit current PROTACs mainly target classically druggable proteins;(2)PROTACs employ cells’ intrinsic protein degradation machinery to destroy target proteins by tagging them and can do so multiple times,therefore it can work at lower dose,in other words,it’s a sub-stoichiometric catalysis;(3)it has few off-target side effects;(4)unlike RNA interference(RNAi)approaches,small-molecule PROTACs have good tissue distribution and oral bioavailability.α1-Adrenergic receptors(α1-ARs),as the crucial members of G protein-coupled receptors(GPCRs),mediate a number of physiological responses of the sympathetic nervous system.So far,α1-ARs have at least three major subtypes(α1A,α1B and α1D).α1-ARs mediate actions of the endogenous catecholamines,norepinephrine,and epinephrine,resulting in smooth muscle contraction,myocardial inotropy and chronotropy,and hepatic glucose metabolism.In addition,some studies have suggested that there is a direct link between α1A-AR stimulation,the activation of transient receptor potential(TRP)-mediated Ca2+ entry,and the proliferation of prostate cancer cells.By using two human prostate cancer epithelial(hPCE)cell models—primary cells from resection specimens(primary hPCE cells)and an LNCaP(lymph node carcinoma of the prostate)cell line,it has been identified thatα1A-AR is functionally coupled to transmembrane Ca2+ entry via the phospholipase C(PLC)-catalyzed inositol phospholipid-breakdown signaling pathway,presumably through activation of channels in the TRP family.This Ca2+ entry seems to be a dominant source of the Ca2+ required to promote the proliferation of LNCaP cells.Therefore,chronic activation of α1A-AR enhanced LNCaP cells proliferation,which could be antagonized by α1A-AR and TRP inhibitors.Collectively,α1A-AR plays an important role in promoting hPCE cells proliferation via TRP channels.Therefore,we can expect more reasonable therapeutic effects if α1A-AR is selectively degraded,which is a novel hypothesis for the therapy of prostate cancer.The main work of in this thesis is to design,synthesis,and biological evaluation of small-molecule PROTACs for α1A-adrenergic receptor.The design of small-molecule PROTACs for α1A-adrenergic receptor is considered mainly from three segments:the ligand of α1A-AR,the ligand of E3 ligase and linker.We chose prazosin as the α1A-AR binding moiety and pomalidomide as CRBN E3 ligase ligand.Therefore,we designed three small-molecule PROTACs(9a-c)by conjugating the key pharmacophore of prazosin as α1A-AR ligand with the pomalidomide as CRBN ligand via PEG linkers with varying length.The target compounds were synthesized via condensation reaction,nucleophilic substitution reaction and Cu(I)-catalyzed alkyne-azide cycloaddition(CuAAC).In the biological evaluation,first of all,we conducted dose-dependent and time-dependent experiments.Among all three PROTACs tested,compound 9c,with the longest PEG linker,was capable of inducing the degradation of α1A-AR(DC50 = 2.86 μM).In addition,compound 9c exhibited potent activity in suppressing the proliferation of PC-3 prostate cancer cells(IC50=6.12 μM).We subsequently carried out an in vivo study to examine the influence of compound 9c on tumor growth.Daily intraperitoneal administration of compound 9c(50 mg/Kg)caused a significant antitumor effect compared with control group(P<0.01).Herein,our small-molecule PROTAC 9c exhibits the in vivo antitumor efficacy,which are also the first small-molecule PROTACs for G protein-coupled receptors(GPCRs)to our knowledge.