This is part 3 of a series of posts delving into the fundamental scientific challenges in drug discovery. Here are the other parts: 1, 2.

Any number of thrillers or action movies should convince us that the first and most important stratagem in defeating an enemy is getting inside his fortress or camp. Once you are inside you still have a long way to go before you accomplish your goals, but you can get nothing done until you breach this very basic first barrier.

It's similar for drugs. Before a drug can cure a little boy's leukemia, before it can restore the cognitive function of an elderly man with dementia, before it can aid the immune system of a firefighter with burns to fight infection, it has to do something very simple - get into cells.

Surprisingly, this process is much more complicated than it appears.

The cell wall is a wonder of evolution, a diverse assembly of biomolecules that is not just a passive barrier to the entry of suspicious molecular cargo but a dynamic microcosm of biochemical activity. As has been known for a century, the wall consists of a lipid bilayer with proteins embedded in it, with many proteins spanning the width of the bilayer. The proteins don't just sit there providing a physical barrier but are constantly at work, acting as molecular machines, moving horizontally and vertically, binding to each other, shuttling cargo inside, often turning inside out in the process. All of them feed on a steady supply of ATP (adenosine triphosphate) for their energy needs.

Not all drugs need to get inside cells - in particular they can sometimes work if they interact with receptor proteins on the cell's exterior - but most of them do. It is the fundamental physiochemical nature of the lipid bilayer that provides the first challenge to drug discovery, and it underscores the multiple variable optimization dilemma that we noted earlier. Here's the problem: there are two kinds of molecules, hydrophobic (oil or lipid-loving) and hydrophilic (water-loving). For a drug to be soluble in water - which bathes the cell and is the major component of the human body - it needs to be hydrophilic, but for it to get past the lipid membrane of the cell it needs to be hydrophobic. These properties are thus orthogonal to each other (not the only time drug designers encounter such a situation), so optimizing one often makes the other worse. If you make a compound too hydrophobic it will likely get past the membrane but it won't dissolve in water to begin with. If you make it too soluble in water it would be too hydrophilic to get past the oily cell membrane. In practice drug designers achieve membrane permeability by striking a balance between these two variables, but predicting this balance is often difficult. Simple measures of hydrophobicity often fail to correlate with membrane permeability. Thus, it's a very fundamental physical property of molecules that poses the first barrier to drugs getting inside cells.

But even if you have a compound that has the right hydrophobicity/hydrophilicity ratio, evolution has conspired to make sure that life still won't be easy for you. And if you think about the way evolution works it makes perfect sense. The cell as a unit arose in a sea of radiation, toxic chemicals and mechanical agitation. It needed strategies to keep out as much material possible, and if this meant occasionally extruding useful molecules like drugs then so be it. The cell also evolved at a time when it had no inkling of life-saving future drugs so there was no reason for it to make it particularly easy for such drugs to enter its innards and perturb its molecular machinery.

Since the cell would rather be safe than sorry, it has evolved a veritable battalion of remarkable proteins whose express purpose is to kick foreign compounds out. The most remarkable among these is a protein whose function would make Sir James (of Dyson vacuum cleaner fame) weep. This protein is called the P-glycoprotein efflux multi-drug transporter (PgP). The fancy name only hides the fact that it's essentially a simple but highly efficient 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. Among many problems, this means that you have to give a higher dose of a drug to a patient than you usually would to account for the dose lost by efflux by PgP, a situation that increases the propensity of side effects.

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 deciphered 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 chains of 1280 amino acids which snake several times through the membrane so that one part of the protein is outside the cell and the other is inside, a mammoth internal cavity of 6000 Å3 and a wondrously complex mechanism of drug binding and extrusion during which the protein undergoes a massive mechanical movement. This movement can be likened to the motion of a revolving door in your favorite mall; the moment a drug enters, PgP makes it do an about turn and kicks it out. 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.

Here's the problem why PgP makes drug discovery so hard: it is practically impossible to predict what kinds of molecules will be potentially pumped out by PgP. Usually drug designers work on the basis of rules of thumb derived from extensive data to narrow down lists of molecules that could evade the lustful embrace of specific proteins; for instance a particular protein causing a side effect may have an overt fondness for benzene rings with a nitrogen in them, another may like positively charged amino acids, yet another may be partial to long, hydrophobic carbon chains. In each of these cases drug designers can then modify the structures of their compounds and get rid of the relevant unwanted chemical appendages that would make them less appetizing to these proteins. But with PgP there is absolutely no clue as to what kinds of molecules it prefers to chew on. PgP is like the world's most obese person, making mincemeat out of anything that comes its way, whether broccoli or cheeseburger. A well-known book on the properties of drugs puts it this way:

"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 (a combination of hydrophobic and hydrophilic)."

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 chemical structure contributing to improved activity - 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.

Ironically 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. Now to be fair, there are also proteins that can actually grab hold of your drug and transport it inside the cell like a benign guard. But - you guessed it - you can't really predict what kinds of molecules these proteins will bind to either.

This concludes the third part of the series on what makes drug discovery so hard. Simply based on fundamental properties it is difficult to engineer properties in a drug that will allow it to get past a cell's membrane. And even if you succeed in achieving this fine balance, there are proteins like PgP embedded in the cell membrane whose express purpose is to throw your drugs out. What is worse is that it has turned out to be very hard to predict exactly what kind of molecules PgP can bind to.

Reason 1: Drugs work by modulating the function of proteins. It’s difficult to find out exactly which proteins are involved in a disease. Even if these proteins are found, it is difficult then to know if their activity can be controlled by a small molecule drug.

Reason 2: Since nature has not really optimized its proteins for binding to drugs, it is very difficult to find a hit for a protein even after searching through millions of molecules, either natural or artificial. And even when a hit is discovered, we don’t know for sure how to turn it into a drug with favorable properties.

Reason 3: It is very difficult to get drugs into cells, partly on the basis of fundamental chemical properties and partly because nature has conspired to create specific proteins whose job is to keep drugs out. What's worse is that we still have little idea exactly what kinds of drugs are kept out by these proteins, so that we have to work on the basis of trial and error to find those that escape their clutches and get in.