transporter – Drug Discovery Opinion

P-Glycoprotein – A Close-up View

P-glycoprotein (Pgp), with its ability to transport a wide range of xenobiotic compounds – including drug molecules – across cell membranes, is the bane of medicinal chemists. Pgp, which probably evolved as a defense mechanism against toxic substances, is an ATP-dependent integral membrane protein that is particularly highly expressed in cells of the gut and kidney, and also in capillary endothelial cells that make up the blood-brain barrier. This means that as well as preventing absorption of drugs from the gut after oral dosing, Pgp can limit entry of drugs into the brain. Expression of Pgp by tumour cells also results in decreased accumulation of anti-cancer drugs in the cells, and contributes to multi-drug resistance in chemotherapy.

A team led by scientists at the Scripps Research Institute have now succeeded in solving the X-ray crystallographic structure of murine Pgp, a result that they hope will help chemists to design more effective drugs. The 3.8Å structure of the apo protein revealed an internal cavity of ca 6000Å³, with a 30Å separation of the two nucleotide-binding domains. Two additional structures with bound inhibitors showed that hydrophobic and aromatic amino acids form distinct drug-binding sites which are capable of stereo-recognition. The apo and drug-bound Pgp structures are open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry, representing initial stages of the transport cycle. The overall structure of Pgp is very similar to that of the bacterial protein, MsbA, which transports lipids out of bacteria, suggesting that Pgp may work in a similar way. In the bacterial transporter, binding of ATP changes the accessibility of the carrier from cytoplasmic (inward) facing to extracellular (outward) facing so that substances caught inside the cavity are ejected from the cell. The cavities of both transporters are lined with hydrophobic amino acids, but Pgp contains a larger number of highly varied amino acids which perhaps explains its broader substrate specificity.

The study, which is published in full in the journal Science, should help chemists to better understand, if not tame, the beast.

New Approach could Starve Tumour Cells to Death

Tumours are heterogeneous and contain both oxygenated and hypoxic regions. Cells in regions with low oxygen levels mainly use glucose for glycolytic energy production and release lactic acid in the process. It had been thought that tumour cells with an ample oxygen supply primarily used glucose for oxidative energy production, but a new study published in the Journal of Clinical Investigation shows that lactate plays a major role in fuelling the oxidative metabolism of these cells. Cells in different regions of a tumour are thus able to mutually regulate their access to energy metabolites, reserving glucose for use by cells in hypoxic regions and recycling their waste product. The study also identified the monocarboxylate transporter 1 (MCT1) as the main route of lactate uptake and, using three different tumour models, showed that blocking MCT1 with α-cyano-4-hydroxycinnamate or siRNA caused a switch from lactate-fuelled respiration to glycolysis. This switch in metabolism of oxygenated cells induces necrosis of distant hypoxic cells by effectively starving them of glucose. These hypoxic cells are known to be very aggressive and difficult to kill with conventional treatments. The reduced oxygen consumption by surviving tumour cells after MCT1 inhibition also rendered the tumours more sensitive to the effects of radiotherapy.

lactic acid Since MCT1 is expressed in a variety of primary human tumours, the study demonstrates the therapeutic potential of MCT1 inhibitors, as well as the likely benefit of combining these with radiotherapy.

Key Role for Bicarbonate Transporter in Anthrax Virulence

Anthrax is caused by the Gram-positive bacterium, Bacillus anthracis. The disease mainly affects herbivorous mammals which ingest or inhale the spores while grazing, but can also be passed to humans by contact with infected animal products. Once within the host, the bacteria begin to multiply and infection typically proves lethal within a few days or weeks. Virulence requires expression of both the anthrax toxin and capsule genes, and one of the first factors found to be important in controlling virulence was elevated levels of CO₂/bicarbonate which are thought to signal the presence of a mammalian host environment. bacillus anthracis It has been difficult to unravel the precise mechanism of virulence control because of the equilibrium between CO₂, H₂CO₃, HCO₃^–, and CO₃^2-, but a study by scientists at the Scripps Research Institute published in the journal PLos Pathogens has demonstrated that expression of a specific bicarbonate transporter is critical for virulence. Deletion of the genes for the transporter strongly decreased the rate of bicarbonate uptake ex vivo and abolished induction of toxin gene expression. Importantly, the strain lacking the transporter was avirulent in a mouse model of anthrax infection, demonstrating the importance of this pathway for recognition of the host environment and pathogenesis.

The identification of an essential bicarbonate transporter may be of relevance to other pathogens, such as Staphylococcus aureus, that also regulate expression of virulence factors in response to CO₂/bicarbonate levels, and suggests a novel target for antibacterial intervention. Similar transporters have been identified and characterized in photosynthetic bacteria, and the availability of 3-dimensional structures of the bicarbonate binding domain of the Synechococcus transporter may help with the design of new inhibitors.

New Brain Delivery Strategy for Parkinson’s Disease

The blood-brain barrier (BBB) fulfills an essential role by restricting the entry of potentially neurotoxic chemicals into brain tissue. The downside of this protective function is that entry of therapeutic molecules into the brain may also be severely restricted; delivering adequate amounts of drugs is one of the biggest challenges in treating many brain diseases.

L-Dopa, used to treat Parkinson’s Disease, is transported into the brain using a carrier system (LAT 1) which normally transports large neutral amino acids. L-Dopa is close enough in structure to one of the endogenous substrates, phenylalanine, to gain entry using this transporter, but the constraints in terms of size and shape on the transported molecule mean that opportunities for such carrier-mediated transport are very limited.

Now Armagen Technologies has announced funding by the Michael J. Fox Foundation for Parkinson’s Research to develop a receptor-mediated system to deliver a neurotrophin into the brain. Receptor-mediated transport mechanisms involve attaching the drug molecule to a protein recognized by cell surface receptors and triggering an energy-dependant transcytosis. In this case, the neurotrophin, which protects the part of the brain that degenerates in Parkinson’s Disease, is fused to a monoclonal antibody which is able to cross the blood brain barrier and so deliver the neurotrophin into the brain tissue.

Receptor-mediated transport mechanisms offer greater flexibility in terms of the size and shape of drug molecules that can be transported, and are likely to be more widely applicable than carrier-mediated systems.