RESUMO
The potential of small molecules to localize within subcellular compartments is rarely explored. To probe this question, we measured the localization of Hsp70 inhibitors using fluorescence microscopy. We found that even closely related analogs had dramatically different distributions, with some residing predominantly in the mitochondria and others in the ER. CRISPRi screens supported this idea, showing that different compounds had distinct chemogenetic interactions with Hsp70s of the ER (HSPA5/BiP) and mitochondria (HSPA9/mortalin) and their co-chaperones. Moreover, localization seemed to determine function, even for molecules with conserved binding sites. Compounds with distinct partitioning have distinct anti-proliferative activity in breast cancer cells compared with anti-viral activity in cellular models of Dengue virus replication, likely because different sets of Hsp70s are required in these processes. These findings highlight the contributions of subcellular partitioning and chemogenetic interactions to small molecule activity, features that are rarely explored during medicinal chemistry campaigns.
Assuntos
Proteínas de Choque Térmico HSP70 , Chaperonas Moleculares , Sítios de Ligação , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Domínios ProteicosRESUMO
Allosteric inhibitors can be more difficult to optimize without an understanding of how their binding influences the conformational motions of the target. Here, we used an integrated computational and experimental approach to probe the molecular mechanism of an allosteric inhibitor of heat shock protein 70 (Hsp70). The anticancer compound, MKT-077, is known to bind a conserved site in members of the Hsp70 family, which favors the ADP-bound state and interferes with a protein-protein interaction (PPI) at long range. However, the binding site does not overlap with either the nucleotide-binding cleft or the PPI contact surface, so its mechanism is unclear. To this end, we modeled Hsp70's internal dynamics and studied how MKT-077 alters local sampling of its allosteric states. The results pointed to a set of concerted motions between five loops in Hsp70's nucleotide-binding domain (NBD), surrounding the MKT-077 binding site. To test this prediction, we mutated key residues and monitored chaperone activities in vitro. Together, the results indicate that MKT-077 interacts with loop222 to favor a pseudo-ADP bound conformer of Hsp70's NBD, even when ATP is present. We used this knowledge to synthesize an analog of MKT-077 that would better prevent motions of loop222 and confirmed that it had improved antiproliferative activity in breast cancer cells. These results provide an example of how to unlock and leverage the complex mechanisms of allosteric inhibitors.
Assuntos
Antineoplásicos/química , Proteínas de Choque Térmico HSC70/química , Piridinas/química , Tiazóis/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Regulação Alostérica , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Cancer cells rely on the chaperone heat shock protein 70 (Hsp70) for survival and proliferation. Recently, benzothiazole rhodacyanines have been shown to bind an allosteric site on Hsp70, interrupting its binding to nucleotide-exchange factors (NEFs) and promoting cell death in breast cancer cell lines. However, proof-of-concept molecules, such as JG-98, have relatively modest potency (EC50 ≈ 0.7-0.4 µM) and are rapidly metabolized in animals. Here, we explored this chemical series through structure- and property-based design of â¼300 analogs, showing that the most potent had >10-fold improved EC50 values (â¼0.05 to 0.03 µM) against two breast cancer cells. Biomarkers and whole genome CRISPRi screens confirmed members of the Hsp70 family as cellular targets. On the basis of these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies support the hypothesis that Hsp70 may be a promising target for anticancer therapeutics.
Assuntos
Benzotiazóis/química , Benzotiazóis/farmacologia , Desenho de Fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Compostos de Piridínio/química , Tiazóis/química , Regulação Alostérica/efeitos dos fármacos , Animais , Benzotiazóis/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP70/química , Humanos , Células MCF-7 , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Normal tau homeostasis is achieved when the synthesis, processing, and degradation of the protein is balanced. Together, the pathways that regulate tau homeostasis ensure that the protein is at the proper levels and that its posttranslational modifications and subcellular localization are appropriately controlled. These pathways include the enzymes responsible for posttranslational modifications, those systems that regulate mRNA splicing, and the molecular chaperones that control tau turnover and its binding to microtubules. In tauopathies, this delicate balance is disturbed. Tau becomes abnormally modified by posttranslational modification, it loses affinity for microtubules, and it accumulates in proteotoxic aggregates. How and why does this imbalance occur? In this review, we discuss how molecular chaperones and other components of the protein homeostasis (e.g., proteostasis) network normally govern tau quality control. We also discuss how aging might reduce the capacity of these systems and how tau mutations might further affect this balance. Finally, we discuss how small-molecule inhibitors are being used to probe and perturb the tau quality-control systems, playing a particularly prominent role in revealing the logic of tau homeostasis. As such, there is now interest in developing these chemical probes into therapeutics, with the goal of restoring normal tau homeostasis to treat disease.
Assuntos
Proteínas de Choque Térmico/metabolismo , Proteostase , Tauopatias/metabolismo , Proteínas tau/metabolismo , Animais , Clusterina/metabolismo , Clusterina/farmacologia , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/farmacologia , Humanos , Mutação de Sentido Incorreto , Ligação Proteica , Tauopatias/tratamento farmacológico , Proteínas tau/antagonistas & inibidoresRESUMO
Heat shock protein 70 (Hsp70) is a chaperone that normally scans the proteome and initiates the turnover of some proteins (termed clients) by linking them to the degradation pathways. This activity is critical to normal protein homeostasis, yet it appears to fail in diseases associated with abnormal protein accumulation. It is not clear why Hsp70 promotes client degradation under some conditions, while sparing that protein under others. Here, we used a combination of chemical biology and genetic strategies to systematically perturb the affinity of Hsp70 for the model client, tau. This approach revealed that tight complexes between Hsp70 and tau were associated with enhanced turnover while transient interactions favored tau retention. These results suggest that client affinity is one important parameter governing Hsp70-mediated quality control.
Assuntos
Benzotiazóis/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Modelos Biológicos , Tauopatias/tratamento farmacológico , Tauopatias/metabolismo , Tiazolidinas/farmacologia , Proteínas tau/metabolismo , Benzotiazóis/química , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP70/química , Células HeLa , Humanos , Estrutura Molecular , Estabilidade Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazolidinas/química , Células Tumorais Cultivadas , Proteínas tau/químicaRESUMO
The rhodacyanine, MKT-077, has anti-proliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogs designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC50 values of 0.4 ± 0.03 µM and 0.7 ± 0.2 µM, respectively). The analogs modestly destabilized the chaperone "clients", Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (t1/2 = 37 min) compared to MKT-077 (t1/2 < 5 min). Finally, NMR titration experiments suggested that these analogs bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogs as chemical probes for studying Hsp70's roles in cancer.
RESUMO
The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of â¼0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.