Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198.

The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC50 of 22.8 nM but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV-VI of CCR5 with an IC50 of 55 nM. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC50 of 122 nM) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC50 of 1023 nM) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile(198).

Physiology and pharmacology of plerixafor.

Autologous hematopoietic stem cell (HSC) transplantation is an important therapeutic option for patients with non-Hodgkin's lymphoma and multiple myeloma. The primary source of HSC is from the peripheral blood which requires mobilization from the bone marrow. Current mobilization regimens include cytokines such as G-CSF and/or chemotherapy. However not all patients mobilize enough HSC to proceed to transplant. The chemokine receptor CXCR4 and its ligand CXCL12 are an integral part of the mechanism of HSC retention in the bone marrow niche. The discovery of plerixafor, a selective inhibitor of CXCR4, has provided a new additional means of mobilizing HSC for autologous transplantation. Plerixafor consists of two cyclam rings with a phenylenebis(methylene) linker. It inhibits CXCL12 binding to CXCR4 and subsequent downstream events including chemotaxis. The molecular interactions of plerixafor have been defined indicating a unique binding mode to CXCR4. Plerixafor rapidly mobilizes HSC within hours compared with the multi-day treatment required by G-CSF in mouse, dog and non-human primate. The mobilized cells once transplanted are capable of timely and endurable engraftment. Additionally CXCR4 has been implicated in the pathology of HIV, inflammatory disease and cancer and the pharmacology of plerixafor in various disease models is described.

Prospective CCR5 small molecule antagonist compound design using a combined mutagenesis/modeling approach.

The viral resistance of marketed antiviral drugs including the emergence of new viral resistance of the only marketed CCR5 entry inhibitor, maraviroc, makes it necessary to develop new CCR5 allosteric inhibitors. A mutagenesis/modeling approach was used (a) to remove the potential hERG liability in an otherwise very promising series of compounds and (b) to design a new class of compounds with an unique mutant fingerprint profile depending on residues in the N-terminus and the extracellular loop 2. On the basis of residues, which were identified by mutagenesis as key interaction sites, binding modes of compounds were derived and utilized for compound design in a prospective manner. The compounds were then synthesized, and in vitro evaluation not only showed that they had good antiviral potency but also fulfilled the requirement of low hERG inhibition, a criterion necessary because a potential approved drug would be administered chronically. This work utilized an interdisciplinary approach including medicinal chemistry, molecular biology, and computational chemistry merging the structural requirements for potency with the requirements of an acceptable in vitro profile for allosteric CCR5 inhibitors. The obtained mutant fingerprint profiles of CCR5 inhibitors were used to translate the CCR5 allosteric binding site into a general pharmacophore, which can be used for discovering new inhibitors.

A time-resolved fluorescent lanthanide (Eu)-GTP binding assay for chemokine receptors as targets in drug discovery.

Chemokines are a family of chemoattractant cytokines involved in leukocyte trafficking, activation, development, and hematopoeisis. Chemokines and their receptors have been implicated in several disease processes, particularly inflammatory and autoimmune disorders and cancer, and are therefore attractive targets for drug development. Chemokine receptors are members of the seven-transmembrane, G protein-coupled receptor (GPCR) family. As such they can be studied using GPCR assays such as ligand binding, G protein activation, and downstream signaling processes such as intracellular calcium flux. In this respect assessing GPCR activation by GTP binding is an important tool to study the early stage of signal transduction. Previously this has been done using the radiolabeled non-hydrolyzable GTP analogue [(35)S]GTPgammaS. In order to avoid the problems involved in working with radioactivity, a new non-radioactive version of the assay has been developed using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence. We have adapted this assay for chemokine receptors. In this chapter, using the chemokine receptor CXCR4 as an example, we describe the steps for assay optimization. In addition we describe adaptation of this assay for the high-throughput screening of chemokine antagonists.

Characterization and Validation of Antibodies for Immunohistochemical Staining of the Chemokine CXCL12.

Chemokines and their receptors have been implicated in cancer biology. The CXCL12/CXCR4 axis is essential for the homing and retention of hematopoietic stem cells in bone marrow niches, and has a significant role in neonatal development. It is also implicated in multiple facets of cancer biology including metastasis, angiogenesis/neo-vasculogenesis, and immune cell trafficking at the tumor microenvironment (TME). Immunohistochemistry (IHC) is an ideal method for investigating involvement of CXCL12 in the TME. Three antibodies were evaluated here for their suitability to stain CXCL12. Both D8G6H and K15C gave apparent specific staining in both lymphoid and tumor tissue, but with converse staining patterns. D8G6H stained cells in the parafollicular zone whereas K15C showed staining of lymphoid cells in the interfollicular zone of tonsil tissue. Using a cell line with high CXCL12 expression, TOV21G, as a positive control, it was found that D8G6H gave strong staining of TOV21G cells whereas no staining was observed with K15C indicating that D8G6H specifically stains CXCL12. Significant staining of CXCL12 in the ovarian TME using tissue microarray was observed using D8G6H. These data demonstrate the importance of antibody characterization for IHC applications, and provide further evidence for the involvement of CXCL12 in ovarian cancer biology.

Comparison of the potential multiple binding modes of bicyclam, monocylam, and noncyclam small-molecule CXC chemokine receptor 4 inhibitors.

CXC chemokine receptor (CXCR)4 is an HIV coreceptor and a chemokine receptor that plays an important role in several physiological and pathological processes, including hematopoiesis, leukocyte homing and trafficking, metastasis, and angiogenesis. This receptor belongs to the class A family of G protein-coupled receptors and is a validated target for the development of a new class of antiretroviral therapeutics. This study compares the interactions of three structurally diverse small-molecule CXCR4 inhibitors with the receptor and is the first report of the molecular interactions of the nonmacrocyclic CXCR4 inhibitor (S)-N'-(1H-benzimidazol-2-ylmethyl)-N'-(5,6,7,8-tetrahydroquinolin-8-yl)butene-1,4-diamine (AMD11070). Fourteen CXCR4 single-site mutants representing amino acid residues that span the entire putative ligand binding pocket were used in this study. These mutants were used in binding studies to examine how each single-site mutation affected the ability of the inhibitors to compete with (125)I-stromal-derived factor-1alpha binding. Our data suggest that these CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor. In addition, our results identified amino acid residues that are involved in unique interactions with two of the CXCR4 inhibitors studied. These data suggest an extended binding pocket in the transmembrane regions close to the second extracellular loop of the receptor. Based on site-directed mutagenesis and molecular modeling, several potential binding modes were proposed for each inhibitor. These mechanistic studies might prove to be useful for the development of future generations of CXCR4 inhibitors with improved clinical pharmacology and safety profiles.

Rhenium inhibitors of cathepsin B (ReO(SYS)X (where Y = S, py; X = Cl, Br, SPhOMe-p)): Synthesis and mechanism of inhibition.

The synthesis of four new oxorhenium(V) complexes containing the "3 + 1" mixed-ligand donor set, ReO(SYS)X (where Y = S, py; X = Cl, Br), is described. All of the complexes tested exhibited selectivity for cathepsin B over K. Most notably, compound 6, ReO(SSS-2,2')Br (IC50(cathepsin B) = 1.0 nM), was 260 times more potent against cathepsin B. It was also discovered that complexes containing the same tridentate (SSS) ligand were more potent when the leaving group was bromide versus chloride (e.g., IC50(cathepsin B): ReO(SSS-2,2')Cl (4), 8.8 nM; ReO(SSS-2,2')Br (6), 1.0 nM). Mechanistic studies with cathepsin B showed that both compounds 2 (ReO(SpyS)(SPhOMe-p)) and 4 were active-site-directed. Compound 2 was determined to be a tight-binding, reversible inhibitor, while compound 4 was a time-dependent, slowly reversible inhibitor. The results described in this paper show that the oxorhenium(V) "3 + 1" complexes are potent, selective inhibitors of cathepsin B and have potential for the treatment of cancer.

Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor, CXCR4.

The chemokine receptor CXCR4 is widely expressed on different cell types, is involved in leukocyte chemotaxis, and is a co-receptor for HIV. AMD3100 has been shown to be a CXCR4 receptor antagonist, and to block HIV infection of T-tropic, X4-using, virus in vitro and in vivo. AMD3100 is an effective mobilizer of hematopoietic stem cells and is being investigated in clinical trials in multiple myeloma and non-Hodgkins lymphoma patients. Using the CCRF-CEM T-cell line that constitutively expresses CXCR4 we confirmed that AMD3100 was an antagonist of SDF-1/CXCL12 ligand binding (IC50=651+/-37 nM). We have also shown that AMD3100 inhibits SDF-1 mediated GTP-binding (IC50=27+/-2.2 nM), SDF-1 mediated calcium flux (IC50=572+/-190 nM), and SDF-1 stimulated chemotaxis (IC50=51+/-17 nM). AMD3100 did not inhibit calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7 when stimulated with their cognate ligands, nor did it inhibit receptor binding of LTB4. AMD3100 did not, on its own, induce a calcium flux in the CCRF-CEM cells, which express multiple GPCRs including CXCR4, CCR4 and CCR7. Furthermore, AMD3100 neither stimulated GTP-binding, an assay for GPCR activation, in CEM cell membranes; nor chemotaxis of CCRF-CEM cells. These data therefore demonstrate that AMD3100 is a specific antagonist of CXCR4, is not cross-reactive with other chemokine receptors, and is not an agonist of CXCR4.

The development of an europium-GTP assay to quantitate chemokine antagonist interactions for CXCR4 and CCR5.

Chemokine receptors have been implicated in several disease processes such as acute and chronic inflammation, cancer, and allograft rejection and are therefore targets for drug development. The chemokine receptors CCR5 and CXCR4 are of particular interest as they serve as entry cofactors for human immunodeficiency virus. These receptors are members of the G protein-coupled receptor (GPCR) family. In this respect, assessing GPCR activation by GTP binding is an important tool to study the early stage of signal transduction. The assay normally utilizes the non-hydrolysable GTP analogue guanosine 5'-gamma-[35S]thiotriphosphate. In order to avoid the problems involved in working with radioactivity, a new non-radioactive version of the assay was developed using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence. The assay was optimized for CXCR4 and CCR5 and validated for screening of chemokine antagonists using the small molecule CXCR4 antagonist AMD3100 and CCR5 antagonists.

The ruthenium-based nitric oxide scavenger, AMD6221, augments cardiovascular responsiveness to noradrenaline in rats with streptozotocin-induced diabetes.

Excess production of nitric oxide by inducible nitric oxide synthase (iNOS) has been implicated in cardiovascular dysfunction associated with the acute phase of diabetes mellitus. We examined if the selective nitric oxide scavenger, AMD6221 (ruthenium[hydrogen(diethylenetrinitrilo)pentaacetato] chloride) improved cardiovascular function in rats with streptozotocin (60 mg/kg, i.v.)-induced diabetes. The cardiovascular effects of noradrenaline (16.5 nmol/kg/min, i.v.) were measured in thiobutabarbitone-anaesthetised diabetic and control rats before and after acute administration of AMD6221 (80 mg/kg). Rats in the acute phase of diabetes (3 weeks post injection of streptozotocin) had impaired mean arterial pressure, left ventricular systolic pressure and maximum rate of increase (+dP/dt) and decrease (-dP/dt) of left ventricular pressure responses to noradrenaline compared with control rats. AMD6221 significantly augmented noradrenaline-induced increases in left ventricular systolic pressure and +/-dP/dt in the diabetic but not control rats. The results show that selective scavenging of nitric oxide by AMD6221 improved cardiac response to noradrenaline in rats with streptozotocin-induced diabetes.

Ruthenium as an effective nitric oxide scavenger.

Whilst nitric oxide (NO) has emerged as one of the most versatile and ubiquitous molecules in the human body with a diverse range of physiological functions, dysfunction in NO biosynthesis or metabolism has led to the pathogenesis of a number of disease states. A variety of therapeutic strategies have therefore emerged that either reduce or increase endogenous NO levels depending on the disease pathology. The predominant strategy to date to reduce levels of NO is to utilise specific isoform selective inhibitors of nitric oxide synthases, the enzymes responsible for NO biosynthesis. An alternative line of attack, not related to specificity for a particular enzyme, but rather on compartmental localisation and pharmacokinetics, is to remove or scavenge the excess NO responsible for the disease pathology. In this regard, a number of NO scavenger molecules have demonstrated pharmacological activity across a broad spectrum of disease states. This review will highlight the rationale behind the development, and the current state of play, of one such class of NO scavengers, complexes of the d-block transition metal ruthenium. Prior to this, a brief overview of the remarkable diversity of NO, both from a chemical and biological viewpoint, will be provided for perspective.

Chemical and Biological Studies of Dichloro(2-((dimethylamino)methyl)phenyl)gold(III).

Several new organogold(III) derivatives of the type [AuX(2)(damp)] (damp = o-C(6)H(4)CH(2)NMe(2)) have been prepared [X = CN, SCN, dtc, or X(2) = tm; dtc = R(2)NCS(2) (R = Me (dmtc) or Et (detc)); tm = SCH(CO(2))CH(2)CO(2)Na] together with [AuCl(tpca)(damp)]Cl (tpca = o-Ph(2)PC(6)H(4)CO(2)H), [Au(dtc)(damp)]Y (Y = Cl, BPh(4)) and K[Au(CN)(3)(damp)]. The (13)C NMR spectra of these and previous derivatives have been fully assigned. In [Au(dtc)(2)(damp)] and K[Au(CN)(3)(damp)], the damp ligand is coordinated only through carbon, as shown by X-ray crystallography and/or NMR. [Au(detc)(2)(damp)] has space group C2/c, with a = 29.884(4) Å, b = 13.446(2) Å, c = 12.401(2) Å, beta = 99.45(3)(o), V = 4915 Å(3), Z = 8, and R = 0.057 for 1918 reflections. The damp and one detc ligand are monodentate, the other detc is bidentate; in solution, the complex shows dynamic behavior, with the detc ligands appearing equivalent. The crystal structure of [Au(dmtc)(damp)]BPh(4) [Pna2(1), a = 26.149(5) Å, b = 11.250(2) Å, c = 11.921(2) Å, V = 3507 Å(3), Z = 4, R = 0.073, 1772 reflections] shows both ligands to be bidentate in the cation, but the two Au-S distances are nonequivalent. The crystal structure of [Au(tm)(damp)] has also been determined [P2(1)/n, a = 18.267(7) Å, b = 9.618(3) Å, c = 18.938(4) Å, beta = 113.45(3)(o), V = 3053 Å(3), Z = 8, R = 0.079, 1389 reflections]. The tm is bound through sulfur and the carboxyl group which allows five-membered ring formation. In all three structures, the trans-influence of the sigma-bonded aryl group is apparent. [AuCl(2)(damp)] has been tested in vitroagainst a range of microbial strains and several human tumor lines, where it displays differential cytotoxicity similar to that of cisplatin. Against the ZR-75-1 human tumor xenograft, both [AuCl(2)(damp)] and cisplatin showed limited activity.

Chemokine receptor modeling: an interdisciplinary approach to drug design.

Chemokines and their receptors are integral components of the immune response, regulating lymphocyte development, homing and trafficking, and playing a key role in the pathophysiology of many diseases. Chemokine receptors have, therefore, become the target for both small-molecule, peptide and antibody therapeutics. Chemokine receptors belong to the family of seven transmembrane receptor class A G protein-coupled receptors. The publication of the crystal structure of the archetypal class A seven transmembrane receptor protein rhodopsin, and other G protein-coupled receptors, including C-X-C chemokine receptor 4 and C-C chemokine receptor 5, provided the opportunity to create homology models of chemokine receptors. In this review, we describe an interdisciplinary approach to chemokine receptor modeling and the utility of this approach for structure-based drug design of chemokine receptor inhibitors.

The molecular pharmacology of AMD11070: an orally bioavailable CXCR4 HIV entry inhibitor.

In order to enter and infect human cells HIV must bind to CD4 in addition to either the CXCR4 or the CCR5 chemokine receptor. AMD11070 was the first orally available small molecule antagonist of CXCR4 to enter the clinic. Herein we report the molecular pharmacology of AMD11070 which is a potent inhibitor of X4 HIV-1 replication and the gp120/CXCR4 interaction. Using the CCRF-CEM T cell line that endogenously expresses CXCR4 we have demonstrated that AMD11070 is an antagonist of SDF-1α ligand binding (IC50 = 12.5 ± 1.3 nM), inhibits SDF-1 mediated calcium flux (IC50 = 9.0 ± 2.0 nM) and SDF-1α mediated activation of the CXCR4 receptor as measured by a Eu-GTP binding assay (IC50 =39.8 ± 2.5 nM) or a [(35)S]-GTPγS binding assay (IC50 =19.0 ± 4.1 nM), and inhibits SDF-1α stimulated chemotaxis (IC50 =19.0 ± 4.0 nM). AMD11070 does not inhibit calcium flux of cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, or ligand binding to CXCR7 and BLT1, demonstrating selectivity for CXCR4. In addition AMD11070 is able to inhibit the SDF-1ß isoform interactions with CXCR4; and N-terminal truncated variants of CXCR4 with equal potency to wild type receptor. Further mechanistic studies indicate that AMD11070 is an allosteric inhibitor of CXCR4.

Strategies for the biological evaluation of gold anticancer agents.

Since the introduction of the monomeric orally bioavailable anti-arthritic gold compound auranofin in 1985, and the success of the platinum-based anti-cancer drugs, there has been a great deal of interest in the use of gold compounds for cancer therapy. However this early promise has not materialized into an approved drug in spite of extensive and innovative efforts in gold chemistry. Therefore, in the light of this lack of success, the strategies for the biological evaluation of potential gold-based anti-cancer drugs are discussed. It is proposed that the biological testing strategy should be multi-faceted incorporating an understanding of the molecular properties of the compounds under investigation related to their behaviour in a biological environment, an evaluation of their comparative in vitro potency against tumor cells, ascertaining the biochemical mechanism of action and target identification to aid in medicinal chemistry design, evaluation of in vivo activity in relevant tumor models, and an understanding of their toxicological and pharmacokinetic properties. This strategy will be exemplified with work on Au(III) cyclometallated complexes in which an integrated approach to the search for new metal-based anticancer drugs was adopted, incorporating in vitro screening, in vivo human tumor xenograft models, and mechanistic studies. The importance of mechanistic studies which have led to the identification of new molecular targets for gold drugs, and in vivo evaluation are emphasized.

Antitumor actions of ruthenium(III)-based nitric oxide scavengers and nitric oxide synthase inhibitors.

The role of endogenous nitric oxide (NO) in the growth and vascularization of a rat carcinosarcoma (P22) has been investigated. Tumor-bearing animals were treated with (i) nitric oxide synthase (NOS) inhibitors, administered via the drinking water, including N(G)-nitro-l-arginine methyl ester (L-NAME), a nonisoform-selective inhibitor, and 2 others that target the inducible (NOS II) enzyme preferentially, namely 1-amino-2-hydroxyguanidine or N-[3-(aminomethyl)benzyl]acetamidine hydrochloride; or (ii) daily injections (intraperitoneally) of 2 Ru(III) polyaminocarboxylates, AMD6221 and AMD6245, both of which are effective NO scavengers. L-NAME, AMD6221, and AMD6245 reduced tumor growth by approximately 60% to 75% of control rates. Tumor sections stained with abs to CD-31/platelet endothelial cell adhesion molecule-1 or NOS III showed that this was associated with a marked reduction (60%-77%) of tumor microvascular densitiy (MVD). Tumors resumed growing promptly when treatment was discontinued, accompanied by partial or complete restoration of MVDs. In contrast, NOS-II selective inhibitors had no effect on tumor growth or vascularization, indicating that both responses require complete blockade of NO production. The results corroborate the view that endogenous NO facilitates tumor development. We suggest that NO deprivation causes tumor feeder vessels to constrict, reducing tumor blood flow. The delivery of oxygen and essential nutrients to the developing tumor is impaired as a consequence, hampering further growth. Normalizing NO levels by withholding treatment causes tumor feeder vessels to dilate, increasing tumor perfusion and reestablishing conditions that allow tumors to begin growing again.

Inhibition of the cathepsin cysteine proteases B and K by square-planar cycloaurated gold(III) compounds and investigation of their anti-cancer activity.

Gold(III) compounds have been examined for potential anti-cancer activity. It is proposed that the molecular targets of these compounds are thiol-containing biological molecules such as the cathepsin cysteine proteases. These enzymes have been implicated in many diseases including cancer. The catalytic mechanism of the cathepsin cysteine proteases is dependent upon a cysteine at the active site which is accessible to the interaction of thiophilic metals such as gold. The synthesis and biological activity of square-planar six-membered cycloaurated Au(III) compounds with a pyridinyl-phenyl linked backbone and two monodentate or one bidentate leaving group is described. Gold(III) cycloaurated compounds were able to inhibit both cathepsins B and K. Structure/activity was investigated by modifications to the pyridinyl-phenyl backbone, and leaving groups. Optimal activity was seen with substitution at the 6 position of the pyridine ring. The reversibility of inhibition was tested by reactivation in the presence of cysteine with a bidentate thiosalicylate compound being an irreversible inhibitor. Five compounds were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. The thiosalicylate compound was tested in vivo against the HT29 human colon tumor xenograft model. A modest decrease in tumor growth was observed compared with the untreated control tumor.

HIV-1 entry inhibition by small-molecule CCR5 antagonists: a combined molecular modeling and mutant study using a high-throughput assay.

Based on the attrition rate of CCR5 small molecule antagonists in the clinic the discovery and development of next generation antagonists with an improved pharmacology and safety profile is necessary. Herein, we describe a combined molecular modeling, CCR5-mediated cell fusion, and receptor site-directed mutagenesis approach to study the molecular interactions of six structurally diverse compounds (aplaviroc, maraviroc, vicriviroc, TAK-779, SCH-C and a benzyloxycarbonyl-aminopiperidin-1-yl-butane derivative) with CCR5, a coreceptor for CCR5-tropic HIV-1 strains. This is the first study using an antifusogenic assay, a model of the interaction of the gp120 envelope protein with CCR5. This assay avoids the use of radioactivity and HIV infection assays, and can be used in a high throughput mode. The assay was validated by comparison with other established CCR5 assays. Given the hydrophobic nature of the binding pocket several binding models are suggested which could prove useful in the rational drug design of new lead compounds.

Cysteine proteases as targets for metal-based drugs.

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.