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.

Pharmacology of AMD3465: a small molecule antagonist of the chemokine receptor CXCR4.

CXCR4 is widely expressed in multiple cell types, and is involved in neonatal development, hematopoiesis, and lymphocyte trafficking and homing. Disruption of the CXCL12/CXCR4 interaction has been implicated in stem cell mobilization. Additionally CXCR4 is a co-receptor for HIV. Selective small molecule antagonists of CXCR4 therefore have therapeutic potential. AMD3465 is an N-pyridinylmethylene monocyclam CXCR4 antagonist which can block infection of T-tropic, CXCR4-using HIV. Using the CCRF-CEM T-cell line which expresses CXCR4 we have demonstrated that AMD3465 is an antagonist of SDF-1 ligand binding (K(i) of 41.7+/-1.2nM), and inhibits SDF-1 mediated signaling as shown by inhibition of GTP binding, calcium flux, and inhibition of chemotaxis. AMD3465 is selective for CXCR4 and does not inhibit chemokine-stimulated calcium flux in cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, nor does it inhibit binding of LTB(4) to its receptor, BLT1. The pharmacokinetics of AMD3465 was investigated in mice and dogs. Absorption was rapid following subcutaneous administration. AMD3465 was cleared from dog plasma in a biphasic manner with a terminal half-life of 1.56-4.63h. Comparison of exposure to the intravenous and subcutaneous doses indicated 100% bioavailability following subcutaneous administration. AMD3465 caused leukocytosis when administered subcutaneously in mice and dogs, with peak mobilization occurring between 0.5 and 1.5h following subcutaneous dosing in mice and with maximum peak plasma concentration of compound preceding peak mobilization in dogs, indicating that AMD3465 has the potential to mobilize hematopoietic stem cells. These data demonstrate the therapeutic potential for the CXCR4 antagonist AMD3465.

Metal compounds for the treatment of parasitic diseases.

The cysteine proteases of the trypanosomatid parasitic protozoa have been validated as targets for chemotherapy of Chagas' disease and leishmaniasis. Metal complexes of gold, platinum, iridium, palladium, rhodium and osmium have been reported to have activity against a variety of trypanosomatids, but the molecular target of these compounds has not been defined. The activity of gold(III) and palladium(II) cyclometallated complexes, and oxorhenium(V) complexes against mammalian and parasitic cysteine proteases was investigated. All gold(III) complexes (1-6) inhibited cathepsin B with IC(50) values in the range of 0.2-1.4 microM. Of the six palladium compounds, aceto[2,6-bis[(butylthio-kappa S)methyl]phenyl-kappa C]-, (SP-4-3)-palladium(II) (11) was the most potent inhibitor of cathepsin B with an IC(50) of 0.4 microM. A clear structure-activity relationship was observed with the oxorhenium(V) complexes with chloro[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium(V) (16) being the most potent inhibitor of cathepsin B with an IC(50) of 0.009 microM. Six complexes were further tested against the parasite cysteine proteases, cruzain from T. cruzi, and cpB from L. major; the most potent inhibitors were the two rhenium complexes (2(1H)-pyridinethionato-kappa S(2))[2,6-bis[(mercapto-kappa S)methyl]pyridine-kappa N(1)] oxorhenium(V) (15) and chloro[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium(V) (16). The compounds were also evaluated in assays for parasite growth. Two oxorhenium(V) compounds ((p-methoxyphenylthiolato-S)[2,6-bis[(mercapto-kappa S)methyl]pyridine-kappa N(1)] oxorhenium(V) (14) and (methanethiolato)[2,2'-(thio-kappa S)bis[ethanethiolato-kappa S)]] oxorhenium (V) (18)) and the palladium compound 11 inhibited T. cruzi intracellular growth, and compound 11 inhibited promastigote growth in three Leishmania species. In conclusion this preliminary data indicates that metal complexes targeted at parasite cysteine proteases show promise for the treatment of both Chagas' disease and leishmaniasis.

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.