Novel natural products as modifiers of multidrug resistance

Abstract

Deoxyspergualin, an immunosuppressive antibiotic, is similar to cyclosporin A and FK506 in some of its properties and mechanisms of action. We therefore decided to examine the effects of deoxyspergualin in multidrug resistant (MDR) cells. Deoxyspergualin did not alter the drug accumulation deficit observed in the MDR, P-glycoprotein-overexpressing cell lines EMT6/AR1.0 or H69/LX4. In addition it was not able to inhibit the ability of [3H]azidopine to photolabel P-glycoprotein. It is unlikely, therefore, that deoxyspergualin is a substrate for P-glycoprotein. Our data suggests that deoxyspergualin is not a good candidate for development as a resistance modifier. Its maintenance of activity in classical MDR cells and its potent in vivo antitumour activity, however, makes deoxyspergualin a promising agent for further investigation into its potential clinical use as an antitumour agent. Several novel fungal products, supplied by Xenova Ltd, were tested for their ability to modify both P-glycoprotein- and MRP-mediated MDR. We demonstrated that, within this series, the most potent modifiers of MDR contained within their structure a tetrahydroisoquinoline group attached to a benzylidene ring in either the meta or paraposition together with the presence of a diketopiperazine ring. The ability of a molecule to be positively charged and lipophilic at physiological pH are also important determinarits of activity as a modifier of P-glycoprotein-mediated MDR. The active compounds inhibit the ability of [3H]azidopine to photolabel P-glycoprotein indicating that they may reverse MDR by themselves binding to P-glycoprotein. Two compounds, XR9006 and XR905I are effective modifiers of P-glycoprotein-mediated MDR. XR9051 is currently undergoing in vivo studies at Xenova. We discovered a novel fungal product modifier of MRP-mediated MDR, XR9173. This compound, synthesised by scientists at Xenova, is cationic at physiological pH and possesses a tetrahydroisoquinoline group. It is also, therefore, an effective modifier of Pglycoprotein-mediated MDR. Recent findings suggest that MRP is identical to the glutathione (GSH) conjugate transporter (Jedlitschky et al, 1994). It is unlikely, however, that XR9I73 reverses MRP-mediated MDR by competing for the same efflux mechanism as GSH as it is not anionic at physiological pH. Our results provide evidence to support the theory that MRP may co-exist with another transporter which is able to transport cationic drugs across the cell membrane and that these transporters are able, in some way, to regulate each others' activity. A potential regulatory mechanism for P-glycoprotein function is modification of phosphorylation. We initially intended to examine the effect of the Xenova compounds on P-clycoprotein phosphorylation. Scientists at Xenova demonstrated that the compounds in this series were not substrates for PKC. It is unlikely, therefore, that they would have any effect on the phosphorylation status of P-glycoprotein. We had already started to examine the effect of several inhibitors of PKC and the PKC activator TPA on the phosphorylauon of P-glycoprotein and on drug transport. The results of these initial studies proved interesting and so we decided to continue with this study. We demonstrated that the PKC inhibitor calphostin C inhibits the phosphorylation of P-glycoprotein and increases dmg accumulation in MDR cells. This indicates that altering P-glycoprotein phosphorylation may indeed alter its function. Interestingly, non-light-activated calphostin C, which does not inhibit PKC, also significantly increased drug accumulation in MDR cells. It appears that other actions of PKC inhibitors with respect to P-glycoprotein, i.e. their ability to bind directly to P-glycoprotein, must also be considered when attempting to correlate effects on P-glycoprotein phosphorylation with effects on drug transport.