IHC staining of Listeria infected mice spleens 6 days after infection with L-monocytogenes, using Anti-Hsp60 (clone: LK2)
Only a few studies have been devoted to develop inhibitors of human Hsp60 (Figure 5). Drugs that are correlated with downregulated levels of Hsp60 include the steroid receptor antagonist mifepristone (RU486) which has been demonstrated to limit the growth of Chlamydophila pneumoniae by binding to and subsequently destroying the bacteria during their normal developmental cycle in epithelial HEp-2 cells 401. Also, enhanced circulating Hsp60 levels as found in HIV patients could be efficiently downregulated in those patients who received combination anti-retroviral therapies 345. In these cases, no direct contact between Hsp60 and the agent became apparent.
Compounds that have been correlated with an overrexpression of Hsp60 comprise bifenthrin 402, bortezomib 403, 1,25-dihydroxyvitamin D3 404, metformin 405, mizoribine 406, and morphine 407. 1,25-dihydroxyvitamin D3 acts as an anti-cancer agent by increasing expression of Fas on the surface of melanoma cells through Hsp60 induction and strengthens caspase sensitivity to Fas-mediated apoptotic pathway by NK cells 404. Proteomic analysis of the rat heart revealed that Hsp60 levels were markedly upregulated after morphine treatment 407. Likewise, exposure of human monocytes to metformin (N,Ndimethylimidodicarbonimidic diamide) led to an upregulation of Hsp60 at both, the mRNA and protein level 405. Metformin is an established oral anti-diabetic drug in the biguanide class representing the first-line drug of choice for the treatment of type 2 diabetes 408. The proteasome inhibitor bortezomib is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. It has been shown previously that treatment of tumor cells with bortezomib led to the upregulation of Hsp60 on the cell surface and promoted their phagocytosis by DCs 403. In a murine ovarian tumor model, tumor-bearing mice treated with bortezomib elicited a CD8+ T cell-mediated inhibition of tumor growth 403.
Mizoribine (Figure 5), a nucleoside-like imidazole-based immunosuppressant which targets the ATPase activity of Hsp60, has been characterized to form a complex with Hsp60 thereby affecting its folding activity 406. Unfortunately, the exact binding site of mizoribine has not been identified although mizoribine has recently been shown to interfere with ATP hydrolysis most likely due to blockage of the dissociation of Hsp10 from the Hsp60/Hsp10 complex 409. It should be noted that this effect could only be observed in the case of the human orthologs. Another Hsp60-targeting compound is the pyrazolo-pyrimidine derivative EC3016, a small molecule inhibitor which specifically inhibits ATPase activity and protein folding by interacting with the ATP binding pocket in Hsp60 (Figure 5) 410.
Epolactaene (Figure 5) belongs to a class of drugs that target critical cysteine residues within Hsp60. Epolactaene inhibits the chaperoning activity of human Hsp60 by covalently binding to Cys442 of human Hsp60 383. The epolactaene derivative ETB (epolactaene tertiary butyl ester; Figure 5) was recently shown to negatively affect the interaction between Hsp60 and the mitochondrial transcription factor Mtf-1 in yeast thereby targeting mitochondrial but not nuclear transcription 411. Analyses of structure/function relationships on epolactaene derivatives led to the identification of the cyclic amide (lactam) and the α,β-unsaturated ketone moieties as being crucially implicated in inhibiting Hsp60 382. Moreover, ETB has been found to suppress hypoxia-induced HIF activation, indicating that Hsp60 affects accumulation of HIF-1α, the oxygen-regulated subunit of HIF-1, directly or indirectly 412. In the same stuidy, the HIF-1α inhibitor carboranylphenoxyacetanilide has been identified to bind primarily to Hsp60. It is interesting to note that Hsp60 has recently been described as being a target of GN26361, a derivative of carboranylphenoxyacetanilide, rendering GN26361 a novel molecular target for HIF-1 inhibition 413, 414.
Regarding the development of Hsp60-targeting drugs, the naturally occurring epolactaene derivative lucilactaene isolated from Fusarium spec. should be mentioned 415. Lucilactaene acts as a cell cycle inhibitor in a p53-dependent manner 416, 417, but its Hsp60-targeting potential has not been elucidated up to date.
The decomposition product of stephacidin B, avrainvillamide (Figure 5) and its derivates are further Hsp60-targeting compounds that have been proposed to alkylate cysteine residues through their electrophilic 3-alkylidene-3H-indole 1-oxide moiety even though the exact interaction site has not been identified up to date 381. Cysteine residues of Hsp60 can also be subjected to sulfation by suvanine, a sulfated tricyclic terpenoid of marine origin harboring anti-inflammatory activities 418. With respect to the redox reactions typically mediated by its cysteine residues, Wang et al. found out that the three critical cysteine residues of Hsp60 (Cys237, Cys442, Cys447) exhibit different responses to the natural Bcl-2 inhibitor (-)-gossypol (Figure 5): an increase of thiol/disulfide ratio for the Cys447 residue due to a decrease of the cellular GSH level, and a decrease of thiol/disulfide ratios for Cys442 and Cys237 residues due to oxidation and sulfation collectively leading to apoptosis induction in ovarian cancer cells after oxidative stress 380. Hsp60 cysteine residues obviously play a pivotal role in binding of the diffusible electrophilic α,β-unsaturated aldehyde 4-hydroxynonenal (HNE) which also targets additional proteins involved in stress signalling pathways 419.
Numerous natural compounds including caryophylene, humulene, sesamol, trans-ferulic acid, vanillyl alcohol, and zerumbone have been reported to differentially reduce Hsp60-mediated cell proliferation of rat arterial smooth muscle cells 420. The inhibitory activity was more pronounced in compounds harboring an 11-membered alicyclic ring favoring the interaction with receptors involved in Hsp60-mediated cell proliferation 420. Despite targeting Hsp60’s ATP binding pocket or cysteine residues, different regions or domains should also be taken into consideration for the development of innovative Hsp60 inhibitors such as the interaction site of Hsp60 and its co-chaperonin Hsp10 or other co-chaperones and client proteins, respectively, critically implicated in cell death pathways.