RESUMEN
PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types, influencing pro-tumor macrophage polarization as well as suppressing the antitumor inflammation response by modulating IFN-γ and IL-4 signaling. PARP14 is a 203â kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates. PARP14 also contains three macrodomains and a WWE domain which are binding modules for mono-ADP-ribose and poly-ADP-ribose, respectively, in addition to two RNA recognition motifs. Catalytic inhibitors of PARP14 have been shown to reverse IL-4 driven pro-tumor gene expression in macrophages, however it is not clear what roles the non-enzymatic biomolecular recognition motifs play in PARP14-driven immunology and inflammation. To further understand this, we have discovered a heterobifunctional small molecule designed based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+ -binding site and recruits cereblon to ubiquitinate it and selectively target it for degradation.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 µM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Inflamación/tratamiento farmacológico , Interleucina-4/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-4/genética , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Poli(ADP-Ribosa) Polimerasas/genética , Células RAW 264.7 , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Interferón Tipo I/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas de Transporte de Nucleósidos/genética , Proteínas de Transporte de Nucleósidos/metabolismo , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Inmunidad Adaptativa/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Escape del Tumor/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.