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1.
Mol Ther ; 31(8): 2408-2421, 2023 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-37408309

RESUMO

Cancer cachexia is a severe systemic wasting disease that negatively affects quality of life and survival in patients with cancer. To date, treating cancer cachexia is still a major unmet clinical need. We recently discovered the destabilization of the AMP-activated protein kinase (AMPK) complex in adipose tissue as a key event in cachexia-related adipose tissue dysfunction and developed an adeno-associated virus (AAV)-based approach to prevent AMPK degradation and prolong cachexia-free survival. Here, we show the development and optimization of a prototypic peptide, Pen-X-ACIP, where the AMPK-stabilizing peptide ACIP is fused to the cell-penetrating peptide moiety penetratin via a propargylic glycine linker to enable late-stage functionalization using click chemistry. Pen-X-ACIP was efficiently taken up by adipocytes, inhibited lipolysis, and restored AMPK signaling. Tissue uptake assays showed a favorable uptake profile into adipose tissue upon intraperitoneal injection. Systemic delivery of Pen-X-ACIP into tumor-bearing animals prevented the progression of cancer cachexia without affecting tumor growth and preserved body weight and adipose tissue mass with no discernable side effects in other peripheral organs, thereby achieving proof of concept. As Pen-X-ACIP also exerted its anti-lipolytic activity in human adipocytes, it now provides a promising platform for further (pre)clinical development toward a novel, first-in-class approach against cancer cachexia.


Assuntos
Proteínas Quinases Ativadas por AMP , Neoplasias , Animais , Humanos , Tecido Adiposo/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Caquexia/tratamento farmacológico , Caquexia/etiologia , Caquexia/metabolismo , Neoplasias/complicações , Neoplasias/metabolismo , Peptídeos/farmacologia , Preparações Farmacêuticas/metabolismo , Qualidade de Vida
2.
Biochem J ; 478(15): 2977-2997, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34259310

RESUMO

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 µM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Benzamidas/farmacologia , Pirimidinas/farmacologia , Proteínas Recombinantes/metabolismo , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Benzamidas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Mutação de Sentido Incorreto , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Pirimidinas/metabolismo , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
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