RESUMEN
SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Antineoplásicos/uso terapéutico , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Macaca fascicularis , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Ratas , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator because of its role in the programmed cell death PD-L1/PD-1 pathway. In the preceding manuscript, we described the optimization of a fused, bicyclic screening hit for potency, selectivity, and physicochemical properties in order to further expand the chemical diversity of allosteric SHP2 inhibitors. In this manuscript, we describe the further expansion of our approach, morphing the fused, bicyclic system into a novel monocyclic pyrimidinone scaffold through our understanding of SAR and use of structure-based design. These studies led to the identification of SHP394 (1), an orally efficacious inhibitor of SHP2, with high lipophilic efficiency, improved potency, and enhanced pharmacokinetic properties. We also report other pyrimidinone analogues with favorable pharmacokinetic and potency profiles. Overall, this work improves upon our previously described allosteric inhibitors and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism.
Asunto(s)
Aminopiridinas/uso terapéutico , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirimidinonas/uso terapéutico , Administración Oral , Regulación Alostérica , Sitio Alostérico , Aminopiridinas/síntesis química , Aminopiridinas/farmacocinética , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Estructura Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Pirimidinonas/síntesis química , Pirimidinonas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos con 2 Anillos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinonas/farmacología , Regulación Alostérica , Sitio Alostérico , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirazoles/síntesis química , Pirazoles/metabolismo , Pirimidinonas/síntesis química , Pirimidinonas/metabolismo , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.
Asunto(s)
Regulación Alostérica , Sitio Alostérico , Piperidinas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirimidinas/farmacología , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Conformación Proteica , Estabilidad ProteicaRESUMEN
The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RASERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RASERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.
Asunto(s)
Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Piperidinas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirimidinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Concentración 50 Inhibidora , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/patología , Proteína Oncogénica p21(ras)/metabolismo , Piperidinas/química , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirimidinas/química , Pirimidinas/uso terapéutico , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.
Asunto(s)
Antineoplásicos/química , Piperidinas/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirazinas/química , Pirimidinas/química , Administración Oral , Regulación Alostérica , Sitio Alostérico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Diseño de Fármacos , Femenino , Xenoinjertos , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Piperidinas/síntesis química , Piperidinas/farmacocinética , Piperidinas/farmacología , Conformación Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Pirazinas/síntesis química , Pirazinas/farmacocinética , Pirazinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.
Asunto(s)
Acetamidas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pirimidinonas/farmacología , Tanquirasas/antagonistas & inhibidores , Acetamidas/administración & dosificación , Acetamidas/química , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Inhibidores Enzimáticos/administración & dosificación , Ratones , Modelos Moleculares , Pirimidinonas/administración & dosificación , Pirimidinonas/química , Relación Estructura-ActividadRESUMEN
Several non-beta-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 DD-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 microM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (Ki = 10 microM) was not influenced by the presence of the substrate.