The chaperone HSP70 promotes the survival of cells exposed to many different types of stresses, and is also potently anti-apoptotic. the enzymatic activity of the anaphase-promoting complex/cyclosome (APC/C). We statement that all three of these compounds can prevent autophagy and cause reduced levels of HSP90 client protein; however, only PES-Cl can prevent the APC/C and induce G2/M arrest. Possible reasons for these differences, and the ramifications for the further development of these ALR prototype compounds as anti-cancer brokers, are discussed. Keywords: phenylethynesulfonamide, PES, PES-Cl, Ver-155008, MKT-077, autophagy, cell cycle, anaphase promoting complex Introduction We previously recognized the compound phenylethynesulfonamide (PES, also known as pifithrin mu) as one that binds specifically to HSP70, and disrupts the ability of this chaperone to interact with crucial co-chaperones that hole to the carboxyl terminus of the substrate-binding domain name of this protein.1 We showed that PES is cytotoxic to tumor cells but not non-transformed cells, and that silencing HSP70 significantly reduces the cytotoxicity of this compound.1 Further, we showed that PES can directly interact with recombinant HSP70, in a manner that is most consistent with a non-covalent association.2 We first became interested in PES as a possible malignancy therapeutic when we discovered that this compound inhibits autophagy, using several different autophagy assays.1 This inhibition of autophagy likely occurs by virtue of the ability of PES to inhibit HSP70 at the lysosome, as there is a concomitant disruption of lysosome function that occurs following PES treatment. This disruption of lysosome function by an HSP70 inhibitor is usually consistent with the findings by others that HSP70 is usually required for lysosome honesty in malignancy cells.3 We also showed that PES can also interact in some malignancy cell lines with the constitutively expressed member buy T16Ainh-A01 of the HSP70 family, HSC70.4 With the knowledge that inhibiting both HSC70 and HSP70 prospects to inhibition of HSP90 chaperone function,5 we investigated and then showed that incubation of cells with PES causes a reduction of HSP90 client protein in the cell; this occurs due to sequestration of HSP90 client proteins into an insoluble portion within the cell.4 Most recently, guided by data indicating that the activity of the anaphase promoting organic/cyclosome (APC/C) requires the function of an ATPase,6 we tested the hypothesis that PES and the related HSP70 inhibitor PES-Cl might inhibit the activity of APC/C in cell-free extracts. We showed buy T16Ainh-A01 that both PES and PES-Cl, but not the HSP90 inhibitor geldanamycin, inhibits the activity of the APC/C in cell-free assays.2 Consistent with this, we found that incubation with PES and PES-Cl causes cell cycle arrest in the G2/M phase of the cell cycle.2 The combined data support the idea that the HSP70 inhibitors PES and PES-Cl possess several notable anti-cancer activities. These include inhibition of autophagy, control of HSP90 client protein solubility, and inhibition of the APC/C. Several groups have previously recognized and characterized other HSP70 inhibitors (for a review observe refs. 7C9). Two of these, VER-155008 and MKT-077, have been well-characterized and are commercially available. The HSP70 inhibitor VER-155008 has been co-crystallized with the HSC70/BAG-1 complex and shown to interact within the ATP-binding pocket of HSC70.10 Like PES, VER-155008 is preferentially cytotoxic to cancer cells but not normal cells, and reduces HSP90 client protein levels in tumor cells.11 The rhodacyanine dye derivative MKT-077 was first discovered as a compound that was cytotoxic to cancer cells but not normal cells, and later shown to bind to the mitochondrial HSP70 member HSPA9 (also called GRP75 or mortalin).12-14 More recently this compound was also found to bind to HSC70, and there is evidence that it can interact with and inhibit HSP70 as well.15 MKT-077 binds near the ATP binding site of HSP70 family members, and alters communication between the nucleotide binding domain name and substrate binding domain name of HSP70, resulting in impaired allostery between these domain names.15 In sum, three different buy T16Ainh-A01 groups have identified three different HSP70 inhibitors, and all three inhibitors show cancer cell-selective cytotoxicity. But all buy T16Ainh-A01 three hole to different regions of HSP70 users (PES and PES-Cl to the substrate-binding domain name, VER-155008 to the ATP binding site, and MKT-077 to an allosteric site near the ATP binding site), and may.