Cysteine proteases as targets for metal-based drugs

Abstract

The discovery of the platinum anticancer drug cisplatin provided a major stimulus for research into metal-based drugs. The molecular target for the platinum agents is DNA; however recent developments in inorganic medicinal chemistry have identified several alternative novel targets for metal-based drugs. Biological molecules with essential thiol groups are attractive targets. Thiol-containing molecular targets include the redox enzymes thioredoxin reductase and glutathione reductase, transcription factors, and cysteine proteases such as caspases and cathepsins. Inorganic chemistry offers many opportunities for medicinal chemistry, and alternative targets for metal-based drugs are reviewed, with a focus on cysteine proteases. The cathepsin cysteine proteases have numerous physiological functions, and have been implicated in diseases including cancer, autoimmune and inflammatory, and parasitic diseases. The catalytic mechanism of these enzymes is dependent upon a cysteine at the active site. We postulate that metal complexes can inhibit these enzymes via a ligand substitution with the thiol of the active site cysteine. We have investigated several classes of metal complexes including cyclometalated organo gold(III) and Pd(II) complexes, and a series of rhenium(V) mixed ligand oxorhenium complexes as inhibitors of cathepsin cysteine proteases. Mechanistic studies were conducted on the latter supporting the hypothesis of active site-directed inhibition. These data are reviewed below and discussed in the context of possible therapeutic applications including cancer and parasitic disease.

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Simon P. Fricker

Simon Fricker is currently a Distinguished Scientific Fellow at Genzyme Corp., USA. He was formerly a member of the Biomedical Technology Group of Johnson Matthey, UK; and subsequently Director, Biology at AnorMED, Canada. His research interests include metal-based drugs, and chemokine receptor biology.

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1. Introduction

The discovery of the platinum-based anticancer drug cisplatin was arguably one of the most significant discoveries in cancer drug development in the twentieth century. Cisplatin, a square planar Pt(II) complex (cis-diamminedichloro platinum(II)) was approved for clinical use in 1978. Platinum drugs are now widely used for the treatment of testicular and ovarian cancers^1,2 and today many chemotherapy regimens contain either cisplatin or another platinum drug. Nephrotoxicity is the dose-limiting toxicity for cisplatin, and this led to research to identify a less toxic agent. These efforts resulted in the discovery and development of the second generation drug carboplatin which was approved in 1986. Since then many laboratories have endeavored to find improved platinum-based drugs and in 2002 oxaliplatin was approved for colorectal cancer. Other agents in development have included the oral agent satraplatin, and picoplatin.^3,4

The molecular mechanism of action of cisplatin is the formation of intrastrand cross-links between neighboring guanine bases on DNA resulting in distortion of the DNA helix. Cell death is associated with the activation of various signal-transduction pathways including those involved in DNA-damage recognition and repair, cell-cycle arrest, and programmed cell death/apoptosis.²

The success of cisplatin and carboplatin stimulated the search for other transition metal-based cancer drugs. A wide range of metals were studied including palladium, iridium, rhodium, ruthenium, tin, copper, and gold.^5,6 Much of the early work on metal-based compounds focused on their interaction with DNA based on the platinum paradigm. However it is now increasingly apparent that many metal complexes with potential for antitumor activity do not behave like cisplatin.^7–11 A series of ruthenium-arene complexes have been designed to interact with DNA in a novel fashion compared to the platinum drugs. These compounds are designed to interact with DNA in a bifunctional manner by both intercalation and direct metalation.¹² Ruthenium complexes such as indazolium [bis-indazoletetrachlororuthenate] (KP1019), and imidazolium [trans-imidazolemethylsulfoxidetetrachlororuthenate], NAMI-A have significantly different properties to cisplatin. The former, which has promising activity against models of colorectal cancer, is redox activated and is transported by the transferrin system.¹³ NAMI-A is only weakly cytotoxic, but interestingly has a profound and selective inhibitory effect on tumor metastasis.⁹

These observations raise the important question of what other molecular targets besides DNA are there for metal-based drugs. A major challenge is how to identify a new molecular target. To a large extent this was ignored in the early years of inorganic medicinal chemistry research where discovery was focused on activity in disease models rather than a molecular target. However, in recent years a more systematic, rational-based drug design approach has become the norm.⁸ This question of novel targets for metal-based drugs is discussed below, with a focus on cysteine proteases.

2. Molecular targets for metal-based drugs

There are several examples where metal-based drugs target biological process other than DNA replication. The superoxide anion, O₂⁻, is a mediator of both acute and chronic inflammation. Under normal circumstances O₂⁻ levels are controlled by the superoxide dismutase (SOD) metalloenzymes. The SOD enzymes are a class of oxidoreductase enzymes containing either copper or manganese at the active site. Small molecule SOD mimetics have been evaluated as therapeutic agents. A Manganese(II) pentaazamacrocyclic complex, M40403, has been shown to have catalytic activity equivalent to the native enzyme, have activity in models of inflammatory disease, and has successfully completed a Phase 1 clinical trial.¹⁴ Nitric oxide is another free radical species involved in normal physiology and in the inflammatory process. Ruthenium(III) polyaminocarboxylates are efficient NO scavengers, being reduced to Ru(II) with formation of a ruthenium-nitrosyl adduct, and have been shown to be active in a variety of disease models.¹⁵

Protein phosphorylation by kinase enzymes, and subsequent dephosphorylation by phosphatase enzymes, is a major regulatory mechanism for protein activity and transmission of intracellular signals. Staurosporine, an indolocarbazole alkaloid, is a relatively non-selective protein kinase inhibitor. Cyclopentadienyl half-sandwich ruthenium complexes are being used as a structural scaffold for staurosporine mimetics. By combining features of the indolocarbazole within the half-sandwich complex highly potent and specific protein kinase inhibitors have been identified for the protein kinases Pim1, MSK1, and glycogen synthase kinase (GSK3α).^16,17 Insulin binds to the insulin receptor activating the Insulin Receptor Tyrosine Kinase (IRTK) triggering a sequence of downstream events. Down-regulation of insulin signaling is mediated by IRTK-associated phosphatases. Oxovanadium compounds, both V(IV) vanadyl, and V(V) vanadate, have been investigated as small molecule insulin mimetics.¹⁸ One of the most extensively studied compounds is bis(maltolato)oxovanadium(IV), also known BMOV. BMOV is orally absorbed, and lowers plasma glucose levels in streptozotocin-induced diabetic rats. An analogue of BMOV, bis(ethylmaltolato)oxovanadium(IV) (BEOV) has completed a Phase I clinical trial. Oxovanadate compounds have been show to exert their insulin-mimetic effect by inhibiting IRTK-associated phosphatases, thus activating the insulin receptor. The situation is more complex with vanadyl compounds. It has been suggested that vandyl may be oxidized to vanadate; alternatively vanadyl may directly stimulate cytosolic protein kinases thus bypassing the insulin receptor. These examples demonstrate that protein phosphorylation can be a target for metal-based drugs.

There has been an ongoing interest in gold compounds for the treatment of cancer. The mechanism of gold antiarthritic drugs is probably multi-factorial, resulting in modulation of the immune response.^19,20 The early promising indication of activity in animal cancer models for gold(I) phosphine compounds, ([Au(dppe)₂]Cl was a lead candidate in preclinical development until it was found to be cardiotoxic) prompted an interest in gold compounds. These initial studies pointed towards DNA as the target for these compounds. Recent work with gold(I) carbene complexes with potential antitumor activity indicate, however, that they target the mitochondria.^21,22 Gold compounds can also inhibit proteases including those involved in proteasomes,²³ and serine proteases such as elastase and cathepsin G.²⁴

Gold(I) compounds have been extensively studied, both for their antiarthritic properties, and as potential anticancer agents.^25,26 In recent years there has been an increasing interest in gold(III) compounds.^26,27 Historically gold(III) has been less well studied because of its reactivity and potential associated toxicity. However, gold(III) has some properties which make it appealing; gold(III) is isoelectronic (d⁸) with platinum(II) and likewise forms square planar complexes and it was therefore tempting to speculate that such complexes would have similar antitumor activity to cisplatin. In collaboration with UMIST, UK, we investigated gold(III) structures which were stabilized with a single mononegative bidentate chelating ligand, 2-[(dimethylamino)methyl]phenyl, (damp).^28,29 This ligand forms part of a 5-membered chelate ring with gold(III) in which the nitrogen of the amine and the carbon of the phenyl ring bond to the metal. The remaining two coordination positions are taken up by either two monodentate ligands (chloride, acetato) or one bidentate ligand (oxalato, malonato, thiosalicylato). These ligands are readily hydrolyzed allowing substitution reactions with biological molecules. These compounds were shown to have selective cytotoxicity across a panel of human tumor cell lines representative of tumors of different tissue origin and clinical response, and to have a modest effect on tumor growth in in vivo tumor xenografts. Investigation of the mechanism of action of the gold(III) complexes indicated that the mechanism was significantly different to their isoelectronic counterparts, the Pt(II) complexes, such as the anticancer drug cisplatin.²⁸ Whereas cisplatin targets DNA it became apparent that this was not the molecular target for the gold(III) complexes. Mechanistic studies focused on the acetato complex. The ability of this compound to interact with DNA was investigated using a number of techniques. The initial studies examined the binding of the compound to a closed circular DNA plasmid, Col E1, by monitoring its mobility on an agarose gel. A DNA binding agent such as cisplatin significantly changes the mobility of the plasmid. The acetato analogue [Au(acetato)₂(damp)] was only able to do this at concentrations up to 1500 times the concentration required to kill tumor cells in vitro. Also, unlike cisplatin, [Au(acetato)₂(damp)] was unable to cross link DNA. Cisplatin causes a block in the cell cycle at the point where DNA repair occurs, the S/G₂ interface, whereas no cell cycle specific effect was noted for [Au(acetato)₂(damp)]. Further supporting evidence came from pharmacological studies. The acetato complex was similarly active against the CH1 and CH1cisR cell line whose mechanism of resistance is repair of cisplatin/DNA lesions. Also [Au(acetato)₂(damp)] had little activity against the mouse ADJ/PC6 tumor, which is known to be particularly sensitive to compounds which cross-link DNA.

Collectively these data point to a different mechanism of action of the Au(III) compounds indicating that the analogy of d⁸, square planar Au(III) and Pt(II) complexes does not extend to their interaction with cells. ¹³C-NMR studies of model reactions with biological ligands showed that the damp complexes have a preference for S-donor ligands such as glutathione and cysteine, with only limited reactivity against nucleosides and their bases.³⁰ We therefore hypothesized that the molecular target for these compounds was a thiol-containing molecule.

Potential thiol-containing molecular targets include caspases (cysteine proteases involved in apopotosis), thioredoxin reductase and glutathione reductase both of which are important in maintaining the redox status of the cell, and transcription factors such as NFκ-B and its regulator IKK, and the cathepsin cysteine proteases. The latter have been implicated in many physiological and pathophysiological processes and thus present a variety of therapeutic opportunities.

3. Cathepsin cysteine proteases as drug targets

Lysosomal cysteine proteases are proteolytic enzymes that are localized in lysosomes and the catalytic activity of which is dependent upon a cysteine residue at the active site.^31,32 This cysteine acts in concert with a histidine, the two together forming a stable thiolate-imidazolium ion pair. Nucleophilic attack by the thiolate on the carbonyl carbon of the peptide bond results in the formation of an acyl intermediate. The collapse of the acyl intermediate results in release of the cleaved peptide and regeneration of the active site (Fig. 1). It was originally thought that the major function of the cathepsin cysteine proteases was a housekeeping role by degradation of proteins taken up into the lysosome. However it is now clear that these proteins are not restricted to localization within the lysosomes but are transported between phagosomes, lysosomes and endosomes, and may accumulate in other organelles under specific physiological conditions. They also can have important roles in processing proteins for example in antigen presentation.³³ In addition cysteine proteases have been implicated in the pathophysiology of several diseases^32,34 including inflammatory airway diseases,³⁵ bone and joint disorders,^36,37 atherosclerosis,^38,39 parasitic diseases,⁴⁰ and cancer^41–44 (Table 1).

Fig. 1 Mechanism of action of cysteine proteases. A stable thiolate-imidazoium ion pair is formed between a histidine (not shown) and a cysteine at the active site. The nucleophilic attack of the thiolate cysteine on the carbonyl carbon on the peptide substrate results in the formation of a tetrahedral intermediate. A covalent-bound acyl enzyme intermediate is formed upon cleavage of the peptide bond. Hydrolysis of the acyl enzyme results in release of the cleaved peptide and regeneration of the enzyme.

Table 1 Cathepsin cysteine proteases as therapeutic targets for disease: relationship between tissue expression, function and disease

Cathepsin	Tissue	Function	Disease
B	Ubiquitous	House keeping	Cancer
			Rheumatoid arthritis
			Osteoarthritis
C	Ubiquitous	Epithelial differentiation	Papillon-Lefevre syndrome
		Immune response
F	Ubiquitous	Antigen presentation	Atherosclerosis
		LDL metabolism
K	Osteoclasts	Bone resorption	Osteoporosis
			Osteoarthritis
			Rheumatoid arthritis
L	Ubiquitous	House keeping	Cancer
		Antigen presentation	Atherosclerosis
		Hair follicle development	Rheumatoid arthritis
			Osteoarthritis
V	Thymus Testis Cornea	Antigen presentation	Autoimmune disease
S	Antigen presenting cells	Antigen presentation	Neurodegenerative diseases
			Rheumatoid arthritis
			Osteoarthritis
			Atherosclerosis

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Cathepsin K is found in osteoclasts and plays a role in bone resorption.^36,45–47 A clear implication of cathepsin K in bone disease came from the observation that mutations in cathepsin K were responsible for the autosomal, recessive bone disorder pycnodysotosis characterized by osteosclerosis and short stature.⁴⁸ Cathepsin K has become a target for bone diseases such as osteoporosis⁴⁹ and the identification of cathepsin K inhibitors has been the focus of intensive efforts by several laboratories in both industry and academia.^50,51

Other cathepsin cysteine proteases have been implicated in disease. Cathepsin S has been shown to be involved in MHC class II-associated invariant chain processing and is the major invariant chain processing enzyme in dendritic cells. Cathepsin S deficient mice have an impaired immune response and show a decreased susceptibility to the development of collagen-induced arthritis.^52,53 Cathepsin S is also unique amongst the cathepsins in that in is active at neutral pH. It is secreted by macrophages and is found in the joints of rheumatoid arthritis (RA) patients, and can degrade proteoglycans.⁵⁴ As such it may be a potential target for drugs for RA.⁵⁵ Cathepsin L also plays a role in antigen presentation and has been implicated in tissue degenerative diseases such as RA, and also atherosclerosis, and cancer. In addition cathepsins S, K and L have been associated with atherosclerosis.^31,32

One of the most extensively studied lysosomal cysteine protease is cathepsin B. Cathepsin B is unique amongst the cathepsin cysteine proteases in that it has both endopeptidase and carboxyexopeptidase activity, whereas other members of this class of proteases are endopeptidases.⁵⁶ This is due to a unique structural feature of cathepsin B, the occluding loop that extends over the active site.⁵⁷ It is believed that a specific histidine, His111, in the occluding loop is responsible for positioning peptide substrates, interacting with the free terminal carboxylate of the peptide substrate, so that cleavage can occur from the C-terminus. This feature potentially provides a means to build in specificity into a molecule for cathepsin B over other cathepsin cysteine proteases.

Cathepsin B is capable of degrading components of the extracellular matrix implicating it in diseases such as muscular dystrophy and rheumatoid arthritis. Cathepsin B expression is located at the outer edge of solid tumors suggesting it is involved in degrading the tumor extracellular matrix thus playing a role in tumor metastasis.⁴³ Increased expression and secretion of cathepsin B have been shown to be associated with numerous human and experimental tumors, and has been proposed to be a prognostic marker for several types of cancer.⁵⁸ The exact role for cathepsin B in solid tumors has yet to be defined, but it has been proposed to be involved in metastasis, angiogenesis, and tumor progression.⁵⁹ Carcinoma cell invasion and metastasis can be inhibited by the nonspecific, irreversible, cysteine protease inhibitor E-64.⁶⁰ Cathepsin B therefore presents itself as a possible therapeutic target for the control of tumor progression.⁶¹

The archetypal cysteine protease inhibitor is E64, a non-selective peptide derivative with an epoxysuccinyl group which irreversibly reacts with the active site cysteine.⁶² The thiol of cysteine has a pK_a of 8.3. However, due to the proximity of the active site histidine the pK_a of the active site cysteine thiol is significantly lower⁶³ and can be can be targeted by electrophilic moieties.⁶² Typical inhibitors contain a peptide portion which recognizes the substrate binding site and can confer specificity to the inhibitor. This portion is commonly referred to as the missile section of the inhibitor. E64 is a non-specific inhibitor. This carries the electrophilic ‘warhead’ which attacks the active site cysteine thiol.³⁶ Several ‘warheads’ have been investigated including aldehydes, semicarbazones, nitriles, cyanamides, ketoheterocycles, ketoamides, vinyl sulfones, and β-lactams.^62,64 The work in our own laboratory has focused on using metal ‘warheads’ to target the active site cysteine.

4. Cathepsin cysteine proteases as targets for metal-based drugs

4.1 Overview

There is now a growing body of evidence to support the concept that biologically active thiol containing molecules are good targets for metal-based drugs. Both gold(I) and gold(III) complexes have been shown to be potent inhibitors of thioredoxin reductase, a putative target for anticancer agents.^22,65,66 There are also a limited number of reports on the inhibition of cysteine proteases by metal complexes. The gold(I) antiarthritic drug auranofin and analogues can inhibit cathepsin B (see below).^67–70 Selenium is an essential element found in selenium-dependent enzymes such as glutathione peroxidase. This prompted the investigation of tellurium(IV) complexes as inhibitors of thiol dependent processes. Organotellurium(IV) complexes were found to be irreversible time-dependent inhibitors of cathepsin B.⁷¹ Ruthenium(II)-arene pta (pta = 1,3,5-triaza-7-phosphaadamantane) compounds (known as RAPTA compounds) are a family of ruthenium(II) compounds of interest as potential antitumor agents that can also inhibit cathepsin B. Molecular docking studies on the RAPTA compounds have shown that inhibitory activity correlates with the size of the substituents on the arene group, with larger substituents resulting in lower activity.⁷² These compounds are reversible time dependent inhibitors, with inhibition being restored by incubation with cysteine. These compounds can also inhibit thioredoxin reductase,⁷² as can the KP1019 analogue trans-[bis(2-amino 5-methylthiazole)tetrachlororuthenate(III)].⁷³

Interestingly we had examined the cytotoxicity of a series of cyclometalated Pd(II) complexes against a panel of human tumor cell lines, originally hypothesizing that the molecular target for these complexes was DNA.⁷⁴ Our recent data has shown that these complexes can inhibit cathepsin B (see below).⁷⁵ Bincoletto et al. have synthesized a cyclopalladated N,N-dimethylamine complex with a bridging bis(diphenylphosphine)ferrocene [Pd₂(C²,N–S₍₋₎dmpa)₂(μ-dppf)Cl₂] with inhibitory activity against cathepsin B.⁷⁶ Kinetic studies indicated a mixed mechanism of inhibition. This compound was also shown to have activity in two rodent cancer models, the Walker-256 mammary carcinoma, and Ehrlich ascites tumor. Perhaps not surprisingly platinum complexes have been investigated as cysteine protease inhibitors, but despite positive results in model reactions they showed no significant inhibitory activity towards cathepsin B.⁷⁷

Our hypothesis was that a metal complex could inhibit a cysteine protease by ligand exchange with the thiol of the active site cysteine. The reduced pK_a of the active site cysteine thiol results in a completely ionic thiolate group which favors the interaction of thiophilic metals such as gold which have a high affinity for thiolates with a low pK_a. In our own laboratory we have investigated several classes of metal complexes including cyclometalated organo gold(III) (Fig. 2) and Pd(II) complexes (Fig. 3), and a series of rhenium(V) mixed ligand oxorhenium complexes (Fig. 4) as inhibitors of cathepsin cysteine proteases. Mechanistic studies were conducted on the latter supporting the hypothesis. These data are reviewed below and discussed in the context of possible therapeutic applications.

Fig. 2 Structures of selected Au(III) complexes. (1) dichloro(2-((2dimethylamino)methyl)phenyl)Au(III), (2) diacetato(2-((2dimethylamino)methyl)phenyl)Au(III), (3) malonato(2-((2dimethylamino)methyl)phenyl)Au(III), (4) dichloro[2-[(2-pyridinyl-κN)methyl]phenyl-κC]Au(III), (5) diaceto[2-[(2-pyridinyl-κN)methyl]phenyl-κC]Au(III), (6) [2-(mercapto-κS)benzoato(2-)-κO)][2-[(2-pyridinyl-κN)methyl]phenyl-κC]Au(III).

Fig. 3 Structures of selected of Pd(II) complexes. (7) chloro(pyridinyl)(2-((2dimethylamino)methyl)phenyl)Pd(II), (8) chloro(isopropylamine)(2-((2dimethylamino)methyl)phenyl)Pd(II), (9) acetato(isopropylamine)(2-((2dimethylamino)methyl)phenyl)Pd(II), (10) aceto[2,6-bis[(methylthio-κS)methyl]phenyl-κC]Pd(II), (11) aceto[2,6-bis[(butylthio-κS)methyl]phenyl-κC]Pd(II), (12) aceto[2,6-bis[(phenylthio-κS)methyl]phenyl-κC]Pd(II).

Fig. 4 Structures of selected oxorhenium(V) ‘3 + 1” complexes. (13) chloro[2,6-bis[(mercapto-κS)methyl]pyridine-κN¹] oxorhenium(V), (14) (p-methoxyphenylthiolato-S)[2,6-bis[(mercapto-κS)methyl]pyridine-κN¹] oxorhenium(V), (15) (2(1H)-pyridinethionato-κS²)[2,6-bis[(mercapto-κS)methyl]pyridine-κN¹] oxorhenium(V), (16) chloro[2,2′-(thio-κS)bis[ethanethiolato-κS)]] oxorhenium(V), (17) (p-methoxyphenylthiolato-S))[2,2′-(thio-κS)bis[ethanethiolato-κS)]] oxorhenium(V), (18) (methanethiolato)[2,2′-(thio-κS)bis[ethanethiolato-κS)]] oxorhenium(V).

4.2 Gold compounds and cancer

The mechanism of action of the disease modifying gold antiarthritis drugs, sodium aurothiomalate, aurothioglucose, and auranofin, has puzzled and perplexed investigators over the years. These drugs, which are all linear gold(I) thiolates, have multiple effects on the immune response and it is probably the broad inhibitory effect, and lack of a specific mechanism of these molecules that accounts for their limited pharmacological effect. Several hypotheses have been advanced including interaction with gene transcription, inhibition of reactive oxygen species, modulation of cytokine levels, and inhibition of bone resorption.^19,78 One interesting hypothesis was that gold compounds may inhibit degradative enzymes including metalloproteases such as collagenase, and cysteine proteases.⁷⁹ Early reports suggested that the level of inhibition was not high enough to account for the therapeutic activity of gold drugs. Recent studies using a higher purity cathepsin B have confirmed that these drugs can inhibit cathepsin B, but with K_i values ranging from 180–440 μM.⁶⁷ Detailed mechanistic studies on auranofin and analogues indicated that the inhibition was both competitive and reversible in the presence of excess thiols.⁶⁸ Inhibition was dependent upon the steric bulk of the phosphine group, and the lability of the leaving group suggesting that inhibition could be improved by appropriate chemical modification of the parent molecule.^69,70

Based on our own mechanistic studies which indicated that the molecular target for gold drugs was likely to be a biological thiol we assayed three cyclometalated gold(III) compounds (compounds 1–3) previously tested for anticancer activity against cathepsin B (Fig. 2) (see section 2). All three inhibited cathepsin B with IC₅₀ values of 0.6–1.36 μM (Table 2).⁷⁵ Attempts were made to make further analogues of these structures. This was found, however, to be synthetically challenging so focus was shifted away from 5-membered chelate rings to 6-membered.⁸⁰ Attempts were made to improve activity by synthesizing a series of six-membered cycloaurated complexes based on a pyridinyl–phenyl linked backbone (compounds 4–6); however no significant improvement in activity was found, with limited relationships between structure and activity. Additionally thiosalicylato complexes were prepared as it was found that the chloro complexes were unstable in plasma. The most notable improvement in activity was seen with substitution at the 6 position of the pyridine ring. As shown in Table 2 the gold compounds 4–6 inhibited cathepsin B but with no marked difference in potency.

Table 2 Inhibition of cathepsin B by gold(III) complexes. Results are expressed as IC₅₀ (concentration of compound giving 50% enzyme inhibition). Errors represent standard error of the mean

Compound	1	2	3	4	5	6
IC50 (μM)	1.36 ± 0.04	0.60 ± 0.04	0.61 ± 0.01	0.85 ± 0.21	1.29 ± 0.09	0.18 ± 0.03

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The reactivity of the complexes towards cysteine was used as a model system to investigate the reactions of the compounds towards thiols. Kinetic studies using UV spectrometry with the dichloro (4) and thiosalicylate (S–O adduct) (6) complexes in the presence of excess cysteine were carried out in an aqueous phosphate buffer (pH = 7.4) at 37 °C. The absorbance changes with time indicated that multiple-step reactions were involved with the presence of excess cysteine for both the dichloro and thiosalicylate adducts.⁸¹ Enzyme kinetic studies with the chloro and acetate complexes indicated that inhibition was time dependent. In addition, this inhibition could be partially reversed by incubation with cysteine indicating that these compounds are tight binding reversible inhibitors (manuscript in preparation).

Selected compounds were tested in an in vitro disease-oriented tumor cell line panel. Different chemosensitivities of the cell lines towards the compounds suggested potential antitumor activity. However, when the thiosalicylate derivative (6), was tested in vivo against the colon HT29 tumor xenograft only modest activity was seen, with no marked improvement over the parent Au(III)damp complexes.⁸² The lack of in vivo activity could in part be explained by the poor solubility and low lipophilicity of these complexes resulting in inadequate biodistribution. Further work is therefore required to produce a target-specific Au(III) drug in this series with a suitable pharmacological activity and toxicity profile. Furthermore the role of cysteine proteases in tumor progression requires further definition, and it is likely that the involvement of cathepsins is different for different tumors and stages of tumor growth.^43,59 Interestingly, thioredoxin reductase has been proposed to be a target for both gold(I) and gold(III) compounds with anticancer activity and this may be a preferred thiol target for gold anticancer drugs.^22,65

4.3 Rhenium(V) oxo compounds and mechanistic studies

The most promising compounds came from a series of rhenium(V) ‘3 + 1’ mixed ligand oxorhenium complexes; where there is one tridentate ligand, and one monodentate ligand.^83,84 The tridentate ligands were either of the type S,S,S or S-pyridyl-S. Rhenium was chosen as rhenium and technetium complexes have been used for tumor imaging and there is therefore a precedent for the clinical use of similar compounds with rhenium compounds having been investigated as imaging agents for brain, heart and tumors.^85–89 The mixed-ligand oxo metal core allows ample opportunities for chemical modification including the coordination of a “receptor-seeking” group thus providing opportunities for chemical modification.⁹⁰ This is particularly relevant from the viewpoint of obtaining a cathepsin B specific inhibitor, our aim being to use this property to target the occluding loop. The monodentate ligand can undergo substitution reactions with thiols, and its reactivity can be modified. Furthermore thiolate ligands because of their π-donating ability can stabilize the five-coordinate ReO³⁺ core. In addition the ability of the monodentate thiolate ligand to be displaced by thiol-containing molecules such as glutathione and cysteine for oxorhenium(V) “3 + 1” complexes suggested their usefulness as potential reversible inhibitors of the cysteine proteases cathepsin B and K.⁸³

A series of rhenium(V) ‘3 + 1’ mixed ligand oxorhenium complexes, examples of which are shown in Fig. 4, were tested for their ability to inhibit cathepsin cysteine protease. These compounds (13–18) were potent inhibitors of cathepsin B, showing selectivity for cathepsin B over cathepsin K. The unique structural feature of cathepsin B, the occluding loop, presents opportunities for building in specificity into the oxorhenium complexes. The histidine residues in the cathepsin B occluding loop, His110 and His111 can undergo hydrogen bonding interactions with H-bond donor groups (e.g. –OH, –NH₃⁺, –CO₂H) of an incoming molecule. With this in mind we hypothesized that the incorporation of a pendant H-bond donor group on the monodentate thiol could interact with the occluding loop histidines thus bringing the electrophilic rhenium center into close proximity with the active site cysteine. In the S,S,S series complexes with monodentate thiol ligands –SR where R is CH₂CH₂CO₂H, CH₂CH₂OH, CH₂CH₂NH₃⁺Cl⁻, or CH₂CH(NH₃⁺)(CO₂⁻) showed a greater than 10-fold selectivity for cathepsin B over cathepsin K.⁸³ In addition the rhenium compounds were inactive against serine protease chymotrypsin indicating specificity for cysteine proteases.⁷⁵

Inhibitory potency for cathepsin B was in part determined by the leaving group with the monochloride complexes (13, 16) being more active than those with a methoxyphenolate leaving group (14, 17) (Table 3). The tridentate ligand also had an effect on activity. For example, comparing complexes with the methoxyphenolate leaving group, the S-pyridyl-S complex (14) was more active than the S,S,S complex (17), presumably due to stabilization of the leaving group by the trans sulfur. The S,S,S chloro complex (16) was the most potent compound. It was hypothesized that this complex adopts a trigonal bipyramidal geometry, readily losing the chloride, to form the preferred square pyramidal geometry upon substitution with the cysteine thiolate at the enzyme active site.⁸³

Table 3 Inhibition of cathepsin B and cathepsin K by oxorhenium(V) complexes. Results are expressed as IC₅₀ (μM concentration of compound giving 50% enzyme inhibition). Errors represent standard error of the mean

Compound	13	14	15	16	17	18
IC50 Cat B	0.90 ± 0.48	6.51 ± 1.20	0.12 ± 0.03	0.0088 ± 0.0002	88.6 ± 6.9	1.26 ± 0.35
IC50 Cat K	4.81 ± 2.35	>50	0.21 ± 0.02	0.40 ± 0.02	>50	23.70 ± 0.95

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Mechanistic studies were performed to test the hypothesis that the compounds were active site inhibitors of cathepsin B. NMR studies of ligand exchange with thiol ligands such as glutathione and 4-methoxythiophenol demonstrated that under conditions in which the incoming ligand is able to exist as an ionized thiolate, ligand exchange reactions were able to take place therefore suggesting that the rhenium complexes should undergo nucleophilic displacement with the highly nucleophilic cysteine residue in the active site of cathepsin B.⁸³ Further studies were performed to investigate the interaction of the two of the compounds [ReO(SpyS)(SPhOMe-p] (14) and [ReO(SSS-2,2′)Cl] (16) with cathepsin B. With standard determinations of enzyme kinetic parameters, active-site titration with inhibitors, and active-site protection with the reversible inhibitor chymostatin, it was demonstrated that both compounds were selective active-site directed inhibitors of cathepsin B.

The epoxysuccinyl inhibitor E64 has been used to titrate the number of active sites of the cathepsin cysteine proteases. As this inhibitor binds irreversibly to the active site cysteine with a 1 : 1 stoichiometry it can be used to estimate the number of enzyme active sites, and hence concentration of activated enzyme, by titrating inhibitor concentration against enzyme activity. Comparative studies with E64 and 16 gave similar estimates of the concentration of active enzyme thus suggesting a 1 : 1 stoichiometry of compound:enzyme active site.

Further indication of the active site-directed nature of inhibition was provided by protection from inactivation studies using a reversible inhibitor of cathepsin B, chymostatin. These studies are based on the assumption that if inactivation by the inhibitor is active site-directed, a known competitive inhibitor will act in the same sub-site of the active site and thereby preventing the test inhibitor from gaining access to the active site resulting in a slower rate of inactivation. Cathepsin B could be protected from inhibition by 16 with addition of the active site directed inhibitor, chymostatin. Both these results indicated that the compounds were directed to the active site. Mass spectrometry studies showed accumulation of a cathepsin B-rhenium complex intermediate for both compounds. The average molecular weight of human cathepsin B was 27823 ± 5 Da (the variability is due to variable glycosylation of the mammalian enzyme). The molecular weight after incubation with either [ReO(SSS-2,2′)Cl] or [ReO(SpyS)(SPhOMe-p] was 27823 + 354 Da and 27823 + 372 Da, respectively, the increase in each case being equivalent to the molecular weight of the rhenium complex less the monodentate ligand. Together these data provide strong evidence of the active-site-directed nature of the inhibition.⁸⁴

Mechanistically, however, there were subtle differences between the two inhibitors which were demonstrated by enzyme kinetic studies. The S-pyridyl-S compound was found to be a tight-binding reversible inhibitor, with its activity being restored in the presence of excess exogenous cysteine, whereas the S,S,S compound was a time-dependent inhibitor, only slowly reversed in the presence of exogenous cysteine. Based upon these data a hypothetical model of compound binding was constructed with the rhenium inhibitor being coordinated to the active site cysteine after ligand substitution of the monodentate ligand (Fig. 5).⁸⁴

Fig. 5 Hypothetical model of the oxorhenium(V) complex 14 binding to the active site cysteine of cathepsin B. (A) Enzyme-inhibitor complex. (B) Close up of the active site with bound inhibitor.

4.4 Parasite cysteine proteases as targets for metal-based drugs

Cysteine proteases have been found to play an integral role in parasitic life cycles including Schistosoma, Plasmodium, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania^91,92 playing multiple roles in the parasite life cycle including nutrition, host invasion, protein processing, and evasion of the host immune response.^93–96 Pathogenic trypanosomatids include Trypanosoma cruzi, the causative agent of Chagas' disease, and the various Leishmania species. Chagas' disease, also know as American trypanosomiasis, is found primarily in South America. Two drugs are available for the treatment of the disease, nifurtimox and benznidazole, and both have associated severe side effects. There is no effective treatment for the chronic form of the disease, whose symptoms include megadisease of the esophagus or colon, and cardiomyopathy, the latter being the most common cause of death. New agents targeted at the sterol biosynthetic pathway are being investigated.⁹⁷ The Leishmanias cause a variety of disease manifestations including cutaneous, mucosal and visceral leishmaniasis, and are found in Central and Southern America, Southern Europe, the Indian subcontinent, the Middle East and Africa. Antimony-based drugs have been the main treatment for cutaneous and visceral disease for over sixty years. Encouraging advances in chemotherapy have been made in recent years. New formulations such as liposomal amphotericin B have been found to be very effective, but its high cost limits its availability to patients. The use of the aminoglycoside, paromomycin, and the alkylphosphocholine, miltefosine, has significantly improved treatment outcomes.^97,98 However there still remains a major need for new drugs for both Chagas' disease and leishmaniasis.

Several metal complexes including platinum(II) and (IV) complexes^99–102 and organometallic complexes of iridium, rhodium, palladium and osmium^102–104 have been reported to have antiparasitic activity against a variety of trypanosomatids. Though there is evidence to suggest that these complexes target organelles such as the nucleus and kinetoplast,^100–104 the molecular target of these compounds has not been defined. In addition gold(I) phosphine and chloroquine complexes have shown activity against trypanosomatids,^79,105–107 and auranofin has shown activity against Plasmodium falciparum and significantly, the proposed molecular target is the thiol-containing enzyme thioredoxin reductase.¹⁰⁸

The cysteine proteases found in T. cruzi and Leishmania have similarities to the mammalian cathepsins B and L. Cruzain is the major cysteine protease found in T. cruzi^109,110 and peptidic inhibitors of cruzain have been shown to be curative in both in vitro and in vivo models of disease.¹¹¹ This has stimulated the search for non-peptidic inhibitors of cruzain.^112,113 There are three gene families of cysteine proteases in the Leishmania species. Disruption of the lmcpb gene array in L. mexicana by gene knockout caused reduced macrophage infectivity.^114,115 However a complete abrogation of infectivity was not seen in an in vivo model of cutaneous leishmaniasis suggesting functional redundancy. Interestingly knockout of both lmca and lmcb genes resulted in a complete eradication of lesions. A pseudopeptide inhibitor of the cathepsin B-like cysteine protease of L. major, cpB, inhibited parasite growth in vitro and ameliorated the pathology associated with a mouse model of leishmaniasis.^116,117 There is therefore an abundance of data to suggest that parasite cysteine proteases represent valid drug targets. We therefore investigated the activity of metal compounds against these cysteine proteases.

We chose to investigate the activity of a selection of the rhenium(V) oxo complexes, gold(III) cyclometalated complexes described above (sections 4.2 and 4.3). Additionally, as we considered that reversible inhibitors were preferable in order to reduce off-target related toxicity we also investigated a series of Pd(II) cyclometalated complexes as palladium complexes are kinetically labile, for example when compared with their Pt(II) counterpart, and are therefore likely to react reversibly with active site thiols. Two cyclometalated palladium(II) complexes, which had been previously tested for anticancer activity in vitro against a panel of human tumor-derived cell lines,⁷⁴ were found to inhibit cathepsin B with IC₅₀ values of 1.52 (7) and 1.71 (8) μM (Table 4).⁷⁵ Structural modifications of the bidentate, 5-membered ring series gave little or no improvement in activity, for example replacing the chloro (7) with an acetate (9) gave an IC₅₀ of 5.0 μM, so, as with the gold series, the focus was shifted to compounds with tridentate ligands and one potential leaving group. In the examples shown in Fig. 3 a thioether group was incorporated into the tridentate ligand to allow for increased chemical diversity. Interestingly modification of this thioether chain gave marked differences in inhibitory potency towards cathepsin B with the butyl thioether (11), being more potent than either the phenyl (12) or methyl (10) analogues (Table 4).⁷⁵

Table 4 Inhibition of cathepsin B by palladium(II) complexes. Results are expressed as IC₅₀ (concentration of compound giving 50% enzyme inhibition). Errors represent standard error of the mean

Compound	7	8	9	10	11	12
IC50 (μM)	1.52 ± 0.22	1.71 ± 0.33	5.00 ± 0.84	13.46 ± 2.34	0.40 ± 0.05	2.74 ± 0.74

---

Six compounds, one gold, one palladium and four rhenium, were selected for studies against the parasite cysteine proteases cruzain from T. cruzi and CPB from L. major. The six compounds had similar rank order potency against the parasite enzymes as against the mammalian cathepsin B (Table 5).⁷⁵ These compounds were further examined in in vitro models of parasite growth and infectivity for T. cruzi and L. major, L. mexicana and L. donovani.

Table 5 Comparison of the inhibitory effect of selected metal compounds against parasite cysteine proteases. Results are expressed as IC₅₀ (μM concentration of compound giving 50% enzyme inhibition). Results for cathepsin B are shown for comparison

Compound	5	11	14	15	16	18
Cat B	1.29	0.40	6.51	0.12	0.0088	1.26
Cruzain	0.7	0.07	0.35	0.015	0.04	5.0
L.major cpB	1.7	2.1	1.0	0.07	0.2	>10

---

The comparative effectiveness of the inhibitors against T.cruzi was estimated from the duration of the intracellular cycle of T. cruzi amastigotes cultured with J774 macrophages as the host cell. The duration of the T. cruzi life cycle in untreated control cells is normally 5 days when infected macrophages die releasing infectious parasites. An increase in macrophage survival is a measure of compound effectiveness. In contrast death of host cells in less than 5 days is indicative of compound toxicity. The compounds were tested at two concentrations of 1 and 10 μM (Fig. 6). The efficacy of the rhenium compounds was limited by their toxicity with both SpyS (14, 15) compounds being toxic to the host cells. Interestingly the methoxy S,S,S complex (18) with the lower enzyme inhibitory activity was active whereas the potent enzyme inhibitor, the chloro S,S,S complex (16) was ineffective. This may be due to the inability of the compound to enter the host cell. The gold compound (5) was both inactive and non-toxic. The palladium compound (11) extended the life cycle at the lower concentration, though toxic at the higher concentration.⁷⁵

Fig. 6 Effect of metal compounds on T. cruzi life cycle. Compounds were tested at 10 μM (gray) or 1 μM (black). Normal life cycle is 5 days, life cycle of <4 days represents toxicity, life cycle >5 days represents inhibition of parasite growth. Two oxorhenium compounds, 14 and 18 were effective at extending the parasite life cycle at 10 μM, and the palladium compound 11 was effective at 1 μM.

Growth inhibition was evaluated for both the extracellular promastigote, and intracellular amastigote stages of the L. major, L. mexicana, and L. Donovani lifecycle. The promastigotes are cultured independent of host cells whereas the amastigotes are cultured with macrophage host cells. Only the palladium compound (11) inhibited promastigote growth completely inhibiting growth at both concentrations.⁷⁵ At the lower 1 μM concentration it was 100% cidal towards L. major and L. mexicana after 4 days, and after 8 days for L. donovani. This is particularly significant as this is the most efficacious compound with reported activity in this assay. None of the compounds was effective against the intracellular amastigote. This may have been due to a combination of a shorter exposure time to the inhibitor (4 days) and the inaccessibility of the Leishmania amastigote within the parasitophorus vacuole within the host cell.

Overall, this data indicates that metal complexes targeted at parasite cysteine proteases show promise for the treatment of Chagas' disease and leishmaniasis. The tridentate cyclometalated Pd(II) complexes show promise as inhibitors of both T. cruzi and Leishmania, and the oxorhenium complexes show promising activity against Leishmania. Given the important role of cysteine proteases in the lifecycle of other parasitic organisms, including Plasmodium, this suggests that there may be potential for metal complexes for the treatment of parasitic disease.

5. Conclusion

The cathepsin cysteine proteases represent potential molecular targets for therapeutic agents.^32,64 In particular cathepsin B and cathepsin K for cancer and osteoporosis respectively show promise. Inhibitors of cathepsin K have received much attention both from academia and the pharmaceutical industry.⁵¹ In addition other enzymes such as cathepsin L and S may be targets for immune-mediated disease.³¹ Parasite cysteine proteases have similarities to mammalian cathepsins and inhibition of these enzymes has been curative in disease models suggesting that these are targets for new agents for these diseases.^89,90

The nucleophilic cysteine thiolate at the cathepsin active site is amenable to attack by metals. There are several reports of metal compounds inhibiting cathepsin B including the gold drug auranofin,^67,68 palladium complexes,⁷⁶ ruthenium arene complexes and organotellurium complexes.⁷¹ We have shown that a variety of metal complexes (Au(III), Pd(II), Re(V)) can be potent inhibitors of cathepsin B, K and the parasite proteases cruzain and cpB.^75,83 Mechanistic studies with oxorhenium complexes have shown that these compounds are active site directed inhibitors.⁸⁴ Inhibitors of the parasite enzymes have shown particular promise in in vitro models of parasitic disease. Both gold(I) and gold(III) compounds have been shown to have potential anticancer activity. Our studies with cyclometalated gold(III) compounds have shown some promise in antitumor models,²⁸ though the extent of the observed activity in vivo has been disappointing so far.⁸²

One of the important findings from our work and that of others is that biologically containing thiol molecules are potentially important targets for metal-based drugs. Several studies provide support for this hypothesis. Transcription factors are central element in the signaling pathway for inflammatory mediators, and as many transcription factors contain cysteine residues they are possible targets for the gold drugs^118,119 and mechanistic studies on the antiarthritic gold drugs have shown that they can inhibit transcription factors. Numerous enzymes catalyzing redox reactions require thiols such as cysteine. These include the flavoenzyme transhydrogenases/disulfide oxidoreductases, and the dehydrogenases. The flavoenzyme transhydrogenases/disulfide oxidoreductases catalyze the reduction of disulfide and are responsible for maintaining the intracellular thiol balance. They contain two active site cysteines and use NADPH and FAD as co-factors. This class of enzymes includes glutathione reductase, thioredoxin reductase, and trypanothione reductase. Elevated levels of thioredoxin can promote cell growth and protect against apoptosis, and have been associated with tumor progression in patients. Inhibitors of thioredoxin reductase have shown activity against tumor cell lines in vitro, and in vivo against human tumor xenografts.¹²⁰ The anticancer activity of both gold(I) and gold(III) complexes has been attributed to inhibition of the redox enzyme thioredoxin reductase.^22,65 Ruthenium complexes such as the RAPTA complexes and ruthenium(III) compounds such as trans-[bis(2-amino 5-methylthiazole)tetrachlororuthenate(III)], and platinum phosphine complexes have also been found to be inhibitors of thioredoxin reductase.^72,73,121

Collectively these observations suggest that the cathepsin cysteine proteases represent potential drug targets for metal-based drugs. Furthermore biological-containing thiol molecules represent an opportunity for the discovery and development of novel metallopharmaceuticals.

Acknowledgements

The author would like to thank and acknowledge the work of the project team who worked on the cysteine protease inhibitors; Beth Cameron, Ian Baird, Yongbao (Bob) Zhu, Jonathan Langille, Micki Olsen, and Renato Skerlj (chemistry), and Renee Mosi, Virginia Annastassov, Jennifer Cox, Gloria Lau, Zefferino Santucci, Rebecca Wong, and Ling Qin (enzymology), and Markus Metz for molecular modeling and Fig. 5. I would also like to thank our collaborators James McKerrow, Elizabeth Hansell and Patricia Doyle from the Tropical Disease Research Unit and Sandler Center for Basic Research in Parasitic Diseases, University of California San Francisco.

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