A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease, and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here, we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360–450 and show that this domain, termed ‘site #1,’ can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher-affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed ‘site #2’ that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity

Chemical-proteomics Identify Peroxiredoxin-1 as an Actionable Target in Triple-negative Breast Cancer

Triple-negative breast cancer (TNBC) is difficult to treat; therefore, the development of drugs directed against its oncogenic vulnerabilities is a desirable goal. Herein, we report the antitumor effects of CM728, a novel quinone-fused oxazepine, against this malignancy. CM728 potently inhibited TNBC cell viability and decreased the growth of MDA-MB-231-induced orthotopic tumors. Furthermore, CM728 exerted a strong synergistic antiproliferative effect with docetaxel in vitro and this combination was more effective than the individual treatments in vivo. Chemical proteomic approaches revealed that CM728 bound to peroxiredoxin-1 (Prdx1), thereby inducing its oxidation. Molecular docking corroborated these findings. CM728 induced oxidative stress and a multi-signal response, including JNK/p38 MAPK activation and STAT3 inhibition. Interestingly, Prdx1 downregulation mimicked these effects. Finally, CM728 led to DNA damage, cell cycle blockage at the S and G2/M phases, and the activation of caspase-dependent apoptosis. Taken together, our results identify a novel compound with antitumoral properties against TNBC. In addition, we describe the mechanism of action of this drug and provide a rationale for the use of Prdx1 inhibitors, such as CM728, alone or in combination with other drugs, for the treatment of TNBC.This project was supported by Centro Atlántico del Medicamento S.A. (CEAMED), CDTI [IDI-20111517], ACIISI [EATIC2017010006], Universidad de Las Palmas de Gran Canaria, Cabildo Insular de Gran Canaria, Fundación del Instituto Canario de Investigación del Cáncer (FICIC), and Miguel Ángel Rodríguez Cardenes. E. Spínola-Lasso is the recipient of a predoctoral fellowship from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información de la Consejería de Economía, Conocimiento y Empleo y por el Fondo Social Europeo (FSE) Programa Operativo Integrado de Canarias 2014-2020, Eje 3 Tema Prioritario 74 (85%) [TESIS2020010081]. J.C. Montero was funded by the Instituto de Salud Carlos III through the Miguel Servet Program [CP12/03073 and CPII17/00015] and received research support from the same institution [PI15/00684 and PI18/00796]. A. Pandiella received funding from the Ministry of Economy and Competitiveness of Spain [BFU2015-71371-R and PID2020-115605RB-I00], Instituto de Salud Carlos III through CIBERONC, Junta de Castilla y León [CSI146P20], CRIS Cancer Foundation, ACMUMA, UCCTA, ALMOM, and the European Community through the Regional Development Funding Program (FEDER)

Structure-activity relationships reveal a 2-furoyloxychalcone as a potent cytotoxic and apoptosis inducer for human U-937 and HL-60 leukaemia cells

Synthetic flavonoids with new substitution patterns have attracted attention as potential anticancer drugs. Here, twelve chalcones were synthesized and their antiproliferative activities against five human tumour cells were evaluated. This series of chalcone derivatives was characterized by the presence of an additional aromatic or heterocyclic ring linked by an ether, in the case of a benzyl radical, or an ester or amide functional group in the case of a furoyl radical. In addition, the influence on cytotoxicity by the presence of one or three methoxy groups or a 2,4-dimethoxy-3-methyl system on the B ring of the chalcone scaffold was also explored. The results revealed that the most cytotoxic chalcones contain a furoyl substituent linked by an ester or an amide through the 2′-hydroxy or the 2′-amino group of the A ring of the chalcone skeleton, with IC50 values between 0.2 ± 0.1 μM and 1.3 ± 0.1 μM against human leukaemia cells. The synthetic chalcone 2′-furoyloxy-4-methoxychalcone (FMC) was, at least, ten-fold more potent than the antineoplastic agent etoposide against U-937 cells and displayed less cytotoxicity against human peripheral blood mononuclear cells. Treatment of U-937 and HL-60 cells with FMC induced cell cycle arrest at the G2-M phase, an increase in the percentage of sub-G1 and annexin-V positive cells, the release of mitochondrial cytochrome c, activation of caspase and poly(ADP-ribose) polymerase cleavage. In addition, it inhibited tubulin polymerization in vitro in a concentration dependent manner. Cell death triggered by this chalcone was decreased by the pan-caspase inhibitor z-VAD-fmk and was dependent of the generation of reactive oxygen species. We conclude that this furoyloxychalcone may be useful in the development of a potential anti-leukaemia strategy.E.S. was supported by the Fundación Instituto Canario de Investigación del Cáncer. E. S.-L. is recipient of a predoctoral fellowship of Consejería de Economía, Conocimiento y Empleo del Gobierno de Canarias in co-financing with Fondo Social Europeo (TESIS2020010081)