Selected Cytotoxic Gold Compounds cause Significant Inhibition of 20S Proteasome Catalytic Activities Nicola Micale, Tanja Schirmeister, Roberta Ettari, Maria A. Cinellu, Laura Maiore, Maria Serratrice, Chiara Gabbiani, Lara Massai, Luigi Messori PII: DOI: Reference:

S0162-0134(14)00221-9 doi: 10.1016/j.jinorgbio.2014.08.001 JIB 9577

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

16 April 2014 31 July 2014 1 August 2014

Please cite this article as: Nicola Micale, Tanja Schirmeister, Roberta Ettari, Maria A. Cinellu, Laura Maiore, Maria Serratrice, Chiara Gabbiani, Lara Massai, Luigi Messori, Selected Cytotoxic Gold Compounds cause Significant Inhibition of 20S Proteasome Catalytic Activities, Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.08.001

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ACCEPTED MANUSCRIPT Selected Cytotoxic Gold Compounds cause Significant Inhibition of 20S Proteasome Catalytic Activities

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Nicola Micale,*a Tanja Schirmeister,b Roberta Ettari,c Maria A. Cinellu,d Laura Maiore,e Maria Serratrice,d Chiara Gabbiani,f Lara Massaig and Luigi Messori*g Department of Drug Sciences and Health Products, University of Messina, Viale Annunziata, 98168 Messina, Italy

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Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, D-55099 Mainz, Germany

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Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20122 Milan, Italy

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Department of Chemistry and Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari, Italy

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Department of Chemical and Geological Sciences, University of Cagliari, S.S. 554, 09042 Monserrato (CA), Italy

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Department of Chemistry and Industrial Chemistry, via Risorgimento 35, 56126 Pisa, Italy

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Laboratory of ‘‘Metals in Medicine’’ (METMED), Department of Chemistry ‘‘Ugo Schiff’’, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy * Corresponding authors: (L. Messori)

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E-mail: [email protected]; Fax: +39 055 4573385; Tel: +39 055 4573388.

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(N. Micale)

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E-mail: [email protected] ; Fax +39 090 676 6402 ; Tel. +39 090 676 6419

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ACCEPTED MANUSCRIPT Abstract Six structurally diverse cytotoxic gold compounds are reported to cause profound and differential inhibition of the three main catalytic activities of purified 20S proteasome while auranofin, an established gold(I) drug in clinical use, is nearly ineffective. In particular, the gold(I) complex [(pbiH)Au(PPh3)]PF6 , turnsout to be the most potent inhibitor of all three enzyme activities with sub-micromolar IC50 values. The present results further support the view that

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proteasome inhibition may play a major – yet not exclusive- role in the cytotoxic actions of gold based anticancer

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agents.

Keywords

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Proteasome; gold compounds; anticancer drugs; enzyme inhibition.

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ACCEPTED MANUSCRIPT The ubiquitin/proteasome system (UPS hereafter) is a complex molecular machinery specifically devoted to the turnover of intracellular proteins in eukaryotic cells; owing to the discovery of UPS and to the assessment of its biological relevance, Hershko and Ciechanover were awarded the 2004 Nobel Prize in Chemistry[1].

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[Insert figure 1]

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The proteasome most exclusively used in mammals is the cytosolic 26S proteasome, ~2 MDa in molecular mass; it contains one 20S core particle capped by two 19S regulatory subunits (Figure 1). The 20S

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core is hollow and forms a cavity where ubiquitin-tagged proteins are degraded. Each end of the core particle associates with a 19S regulatory subunit containing multiple ATPase sites and ubiquitin binding sites; this structure is capable of recognising poly-ubiquitinated proteins that are then transferred to the catalytic interior[2]. Three main enzyme activities were identified in the proteasome, namely the chymotryptic-like (CT-

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L), the caspase-like (C-L; also known as post glutamyl-peptide hydrolyzing, PGPH) and the tryptic-like (T-L). These enzymatic activities are performed by distinct inner subunits (β5, β2, and β1, respectively) characterised

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by N-terminal catalytic threonine residues [3,4]. The CT-L activity is considered the most important one; its inhibition is commonly associated to relevant pro-apoptotic and antiproliferative effects. Targeting the CT-L active site has long been considered as sufficient to develop new candidate drugs for cancer treatment; yet, inhibition of multiple active sites is usually required to decrease markedly protein degradation and produce

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more relevant biological effects [5]. So, beyond the β5 active site, it is also important to target either the β1 or

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β2 active sites, considered as co-targets of cancer drugs [6,7,8]. Specific assays were developed to monitor independently the three individual catalytic activities of the proteasome and a number of selective inhibitors were identified [9,10,11].

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The UPS-dependent degradation pathway plays an essential role both in up-regulation of cell proliferation and down-regulation of cell death in human cancer cells and represents a validated druggable target. In vitro and in vivo experimental and clinical results clearly documented the use of proteasome inhibitors as potential anticancer drugs [12]. Proteasome inhibition in cancer cells leads to accumulation of pro-apoptotic proteins

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followed by induction of cell death [13]. The clinical efficacy of the proteasome inhibitor bortezomib toward multiple myeloma and other haematological malignancies provided the conclusive "proof of concept" that targeting the proteasome is a feasible and innovative strategy in cancer treatment [14]. Currently, there is a great interest for the discovery of new and structurally diverse anti-proteasome agents, unrelated to bortezomib. Among them, a few metal-based compounds were reported to behave as effective proteasome inhibitors [15,16]; in particular, the cytotoxic gold(III) dithiocarbamate compound AuL12 was shwon to cause remarkable proteasome inhibition. A rather strong inhibition of CT-L activity of 20S proteasome was documented in vitro for AuL12 with IC50 values ~1.1 μM [17]. Subsequent studies in animal models confirmed the favourable anticancer properties of this gold(III) complex; in particular, AuL12 turned out very promising for treatment of breast and prostate cancer [18]. Prompted by these arguments, we decided to investigate whether a panel of six structurally diverse gold compounds, available in our laboratories, might act as effective inhibitors of UPS.

The compounds that were chosen for the present investigation are synoptically represented in Chart 1. Notably, this panel comprises two mononuclear gold(III) complexes, K[Au(Sac) 3Cl] [19] and (pbi)Au(OAc)2,[20], an organogold(III) compound, Aubipyc,[21], two dinuclear gold(III) complexes,

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ACCEPTED MANUSCRIPT Auoxo6[22] and Au2phen,[23,24], a gold(I) phosphine complex, [(pbiH)Au(PPh3)]PF6 [20]. Auranofin, an established gold drug in clinical use for rheumatoid arthritis treatment, was also included in the panel for reference purposes. Thus, this panel of metal complexes features the gold center within a variety of redox states and coordination environments. Notably, the antiproliferative properties of all these gold compounds had been previously measured in vitro toward the representative A2780 human ovarian carcinoma cell line (see

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Table 1, last column) [19,20,21,22,24,25].

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[Insert chart 1]

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The CT-L, T-L and C-L enzyme activities of 20S proteasome were systematically and independently assayed upon addition of each gold compound. Details of the assays used to monitor the three distinct catalytic activities are provided in the Experimental Section. The selectivity of these compounds towards the target enzyme was determined with the same method by using bovine pancreatic α-chymotrypsin, cathepsin-B and

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cathepsin-L. For comparison purposes parallel experiments were carried out on cisplatin [26] and NAMI A, [27,28] two representative metal-based anticancer drugs, respectively containing platinum(II) and

enzyme inhibition are reported in Table 1.

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ruthenium(III), but both failed to produce any significant enzyme inhibition. All relevant results concerning

On the whole, all six gold compounds (b-g) turned out to cause a very conspicuous inhibition of proteasome CT-L activity, with IC50 values often comparable and in one case lower than the value of 1.1 μM

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previously determined for AuL12 (h)[17]. Noteworthy, all panel compounds also resulted quite selective in the

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test against bovine pancreatic α-chymotrypsin with the only exception of [(pbiH)Au(PPh 3)]PF6 (f); in any case even the latter afforded a rather modest inhibition (see Table 1, penultimate column). Unfortunately, the same degree of selectivity was not detected in the case of cysteine proteases (i.e. cathepsins) indicating a certain

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affinity of these gold complexes for thiol groups. The percentage of inhibition ranged from 56% to 95% when screened at 20 µM concentrations, with the sole exception of Auranofin that exhibited a value 100

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0.5 [25]

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3.0

0.94

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3.3 [21]

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1.3

3.1

1.9

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1.79 [22]

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1.2

1.9

1.9

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0.8 [24]

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6.3

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23.6 [19]

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0.89

0.76

0.078

20.3

1.5 [20]

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1.4

77.0

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1.90 [20]

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CT-L

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Enzyme inhibition data(µM) and antiproliferative activities (IC50 µM) for the various gold compounds

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ACCEPTED MANUSCRIPT CAPTION TO THE FIGURES

Chart 1. Schematic representation of panel compounds.

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(a) Auranofin; (b) Aubipyc; (c) Auoxo6; (d) Au2phen; (e) K[Au(Sac)3Cl]; (f) [(pbiH)Au(PPh3)]PF6; (g)

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(pbi)Au(OAc)2; (h) AuL12 (previously tested)

Fig. 1. Scheme of 26S proteasome.

(a) The 26S proteasome is a 2MDa protein complex containing one 20S core (gray) and two regulatory 19S

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subunits (white); (b) Bortezomib interacts with threonine residues located on the β5 subunit that confers

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chymotryptic proteolytic activity.(reproduced from Zavrski, I. et al.; Anti-Cancer Drugs. 2005, 16, 475-481).

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Chart 1.

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Fig.1.

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Pictogram for the graphical abstract

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ACCEPTED MANUSCRIPT

Synopsis for the graphical abstract

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The 20S core of proteasome is constituted of two outer rings, composed of seven different α-subunits which serve as an anchor for the 19S regulators and two inner rings, consisting of seven different β-subunits, which contain the three different catalytic sites (caspase-like, trypsin-like, and chymotrypsin-like sites).

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ACCEPTED MANUSCRIPT

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Highlights

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- Various cytotoxic gold compounds behave as effective inhibitors of 20S proteasome.

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- [(pbiH)Au(PPh3)]PF6 inhibits all three enzyme activities of 20S proteasome

- Proteasome inhibition may be involved in cytotoxic mechanism of several gold compounds

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- Gold(I) coordination to 20S proteasome’ site seems to be the crucial event for enzyme inhibition

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