RESUMO
Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.
Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Ácidos Nipecóticos/química , Ácidos Nipecóticos/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Benzimidazóis/metabolismo , Benzimidazóis/farmacocinética , Disponibilidade Biológica , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Humanos , Camundongos , Modelos Moleculares , Ácidos Nipecóticos/metabolismo , Ácidos Nipecóticos/farmacocinética , Permeabilidade , Piperidinas/metabolismo , Piperidinas/farmacocinética , Agregados Proteicos/efeitos dos fármacos , Estrutura Terciária de Proteína , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85(ErbB2)) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85(ErbB2). Here we show that PUVA reduced p85(ErbB2) phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.