RESUMO
We report herein the discovery of highly potent PROTAC degraders of androgen receptor (AR), as exemplified by compound 34 (ARD-69). ARD-69 induces degradation of AR protein in AR-positive prostate cancer cell lines in a dose- and time-dependent manner. ARD-69 achieves DC50 values of 0.86, 0.76, and 10.4 nM in LNCaP, VCaP, and 22Rv1 AR+ prostate cancer cell lines, respectively. ARD-69 is capable of reducing the AR protein level by >95% in these prostate cancer cell lines and effectively suppressing AR-regulated gene expression. ARD-69 potently inhibits cell growth in these AR-positive prostate cancer cell lines and is >100 times more potent than AR antagonists. A single dose of ARD-69 effectively reduces the level of AR protein in xenograft tumor tissue in mice. Further optimization of ARD-69 may ultimately lead to a new therapy for AR+, castration-resistant prostate cancer.
Assuntos
Antagonistas de Receptores de Andrógenos/química , Proteólise , Receptores Androgênicos/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Proteólise/efeitos dos fármacos , Receptores Androgênicos/genética , Relação Estrutura-Atividade , Transplante Heterólogo , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Beyond the targeting of E3 ubiquitin ligases to inhibit protein homeostasis, E3 ligase binders can be repurposed as targeted protein degraders (PROTACs or molecular glues). We sought to identify new binders of the VHL E3 ligase by biophysical fragment-based screening followed by X-ray crystallographic soaking. We identified fragments binding at the ElonginC:Cullin2 interface and a new cryptic pocket in VHL, along with other potential ligandable sites predicted computationally and found to bind solvent molecules in crystal structures. The elucidated interactions provide starting points for future ligand development.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Complexos Multiproteicos/química , Ubiquitina-Proteína Ligases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Elonguina/química , Elonguina/metabolismo , Fluorometria/métodos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/metabolismo , Policitemia/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/química , Proteína Supressora de Tumor Von Hippel-Lindau/químicaRESUMO
A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.
Assuntos
Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Proteínas Virais/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Aminoácidos/química , DNA/química , Avaliação Pré-Clínica de Medicamentos/métodos , Terapia de Alvo Molecular , Peptídeos Cíclicos/genética , Reação em Cadeia da Polimerase , Vírus Sinciciais Respiratórios , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteína Supressora de Tumor Von Hippel-Lindau/químicaRESUMO
The design of proteolysis-targeting chimeras (PROTACs) is a powerful small-molecule approach for inducing protein degradation. PROTACs conjugate a target warhead to an E3 ubiquitin ligase ligand via a linker. Here we examined the impact of derivatizing two different BET bromodomain inhibitors, triazolodiazepine JQ1 and the more potent tetrahydroquinoline I-BET726, via distinct exit vectors, using different polyethylene glycol linkers to VHL ligand VH032. Triazolodiazepine PROTACs exhibited positive cooperativities of ternary complex formation and were more potent degraders than tetrahydroquinoline compounds, which showed negative cooperativities instead. Marked dependency on linker length was observed for BET-degrading and cMyc-driven antiproliferative activities in acute myeloid leukemia cell lines. This work exemplifies as a cautionary tale how a more potent inhibitor does not necessarily generate more potent PROTACs and underscores the key roles played by the conjugation. The provided insights and framework for structure-activity relationships of bivalent degraders are anticipated to have wide future applicability.