P-glycoprotein: The vacuum cleaner that makes Sir James weep

There has been a lot of discussion during the last decade about the continuing attrition in the pharmaceutical industry and the absence of novel drugs. Several factors including layoffs, narrow-minded management practices, outsourcing etc. have been held responsible for this trend which only promises to exacerbate in the near future. All eminently sensible points. But one thing should be clear; drug discovery remains hard because we still just don't understand a lot of the basic science very well. This is something that should always be on the mind of anyone who wants to hold  non-scientific factors responsible for drug failures. The fact is that there still remain very basic challenges that drug discoverers have to surmount. And by scientific challenges I am not talking about cutting edge, futuristic, overhyped strategies like gene therapy and nanotechnology that haven't yet borne fruit. I am talking about fundamental challenges here, problems that have been realized for years and yet not solved.

A review in this week's issue of Journal of Medicinal Chemistry has an account of something that greatly contributes to one of these challenges; getting compounds into cells. It's a basic problem in developing any drug. You may have a molecule that looks miraculous in the test tube but which utterly fails once you put it into a living organism. Many factors can contribute to this lack of translation but one of the most basic reasons is simply that the compound is not getting inside the cell. Recall that the cell membrane is expressly designed to keep things out, which is a good thing for evolution but a bad thing for drug designers. The membrane is composed of phospholipids with all kinds of proteins and other biomolecules embedded within it. Drugs can get across this membrane by simple passive diffusion, although in some cases they may be shuttled across by special helper proteins. In general any foreign substance will have to be hydrophobic enough to get past this membrane. But even otherwise it will have to satisfy some simple properties; it can't be too big and charged for instance. And it can't be too hydrophobic otherwise it won't dissolve in the aqueous medium surrounding the membrane in the first place.

But hydrophobicity is where your troubles only begin. Cells have an assortment of watchdog proteins whose purpose is to keep out unwanted substances. In the modern world, "unwanted substances" includes pretty much all drugs. The J. Med. Chem. review focuses on one of these watchdogs - very likely their king - which plagues drug designers all the time; the P-glycoprotein efflux multi-drug transporter (Pg). The fancy name only hides the fact that it's essentially a simple pump embedded within the membrane, designed to throw drugs out. It's the ultimate bouncer; even drugs that have the right mix of hydrophobic and hydrophilic character quake and rapidly exit when they encounter PgP. In fact the protein was discovered when it was found that some cancers were becoming resistant to certain drugs; what was happening was that these drugs were being pumped out or "effluxed". Even worse, the presence of these drugs was increasing the expression of the protein. Later it was found that a wide variety of drugs bind to and increase the expression of Pgp, reducing their effective concentration inside the cell; it's still one of the principal mechanisms of resistance in some kinds of cancer. 

Progress was only hindered by not knowing the structure of the protein (a part of which is illustrated above) which was only recently and partially solved by x-ray crystallography, and even then it's not really helping. The protein's structure and interior are exquisitely hideous to say the least; 12 transmembrane segments composed of 1280 amino acids, a mammoth internal cavity of 6000 Å³ and a wondrously complex mechanism of compound binding and extrusion during which the protein undergoes a massive conformational challenge. As it snakes its way through the lipid bilayer and wraps itself around drugs, the precision of this molecular machine would be wholly admirable if it were not for the eminent heartburn that it causes drug discoverers. 

The constant extrusion of drugs by Pgp means that you may have to increase the dosage of your drugs (or saturate the protein with another drug) to maintain high blood levels, but that's just skimming the surface of the Pgp world of pain. Since its original discovery the protein has turned into a minor nemesis for drug designers and it's become a part of a notorious list of proteins called "anti targets" that can lead to side-effects and lack of efficacy (we encountered one of these anti targets before - the hERG channel protein). And that's not only because Pgp is ubiquitously expressed in the intestine and liver where most drugs are metabolized. Nor is it because of its special role in the blood-brain barrier which creates additional problems for CNS drugs. It's because when it comes to Pgp, scientists may not have a clue about how to possibly solve the problem. Usually when you encounter an unwanted protein that binds to your drugs, you try to add a modification to your drug to block this binding. In many cases, structure-activity relationship (SAR) can help you pin down some trends; you remove a basic nitrogen atom here, you get rid of a double bond there, you add a fluorine to that ring. If you know what kinds of molecular features a rogue antitarget protein likes, you can avoid those features in your drug.

But not so for Pgp. Pgp is, in the words of the review author, a "hydrophobic vacuum cleaner". And it's one that will put Sir James to shame. What kinds of molecules does it like as substrates? Here's a description from Kerns and Di's book "Drug-like Properties":

"The substrate specificity for Pgp is very broad. Compounds ranging from a molecular weight of 250 to 1850 are known to be transported by Pgp. Substrates may be aromatic, non-aromatic, linear or circular. They can be basic, acidic, zwitterionic or uncharged. Some substrates are hydrophobic, others are hydrophilic and yet others are amphipathic."

The authors could have saved themselves all those words by simply saying something like "Pgp binds to and extrudes everything in the universe except possibly the human soul". As should be obvious, this kitchen sink description of every molecule of every kind is not exactly a guide for drug designers to rationally add modifications that would prevent Pgp binding. I was myself part of a project where the whole "rational" drug design process was going extremely well - well-defined changes in structure contributing to improved potency - until we found that the compounds were being generously ejected by Pgp. At this point our gung-ho approach screeched to a halt and we found ourselves transported from the sunlight of rational design into the night of Pgp-mediated chaos. Where before we had been confidently stepping across a brightly lit landscape, we now found ourselves groping around in the dark with our eyes closed. It was like falling down an abyss. There was no rational modification to our existing molecules that would ensure a Pgp-free existence. From then on it was largely about gut feelings, intuition and Hail Mary passes.

In reality, Pgp binding is sometimes considered so painfully complex to circumvent that the best strategy may actually be to wave a wand and temporarily forget about it. Counterintuitive as this seems, what this strategy means is that often the best way to prevent Pgp drug binding is to simply increase the passive diffusion of your compounds so much that it swamps any Pgp-enabled extrusion. Basically you just keep on bumping up the magnitude of one process until it can one-up the opposing process.

The present review in J. Med. Chem. provides some respite from this depressing existence. The author describes several case studies where strategies like tying up hydrogen bond donors, getting rid of them, reducing basicity or reducing polar surface area helped to design out Pgp binding. These are valuable examples, but they are anecdotal nonetheless and may not work for other molecules with similar functionalities. Well-defined rational approaches to Pgp binding are still lacking and the complex mechanism of the Pgp-drug binding precludes designing specific Pgp inhibitors even if the structure is known. Reviews like the present one provide useful guidelines, but for the foreseeable future at least, Pgp will stand in splendor as one among a handful of scientific challenges that continue to make drug discovery just so damn difficult.
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