Innovations in modern medicine have made many previously incurable diseases easily treatable over the past few decades, which has had a huge impact on public health. Significant breakthroughs include the discovery of insulin, which can be used to treat diabetes. However, drug discovery and development is a complex process as it typically takes about 12 to 15 years and costs more than two billion US dollars to bring a new drug to the market.
One of the major hurdles is that the new drug needs to travel to target tissues in the body to carry out its activities. More specifically, the concentration of drugs in the target site needs to reach the threshold level in order to optimize drug efficacy. Meanwhile, the body needs to avoid the accumulation of excessive amounts of drugs in other tissues to minimize drug toxicity. So, how does the body balance drug concentration to give optimal efficacy while maintaining minimal toxicity?
Drug transporters play key roles in achieving optimal drug concentration by uptaking drugs into target cells and exporting excessive drugs out of the cells. They are essential for the pharmacokinetics of drugs including their absorption, distribution, metabolism, and excretion (ADME). And importantly, drug transporters are membrane proteins, which form part of the cell membrane.
The structural classification of membrane proteins
Membrane proteins are proteins that are either a part of or interact with the biological membranes. Interestingly, membrane proteins account for about a third of human proteins and they fall into two major categories based on the nature of the protein-membrane interactions: integral membrane proteins and peripheral membrane proteins.
Integral membrane proteins are permanently anchored to the biological membrane, which consists of a lipid bilayer. They can be classified into integral polytopic proteins, also known as transmembrane proteins, which span across the bilayer; integral monotopic proteins that only associate with one side of the membrane; or lipid-anchored proteins, which associate with the membrane via a lipid group such as a fatty acid. Peripheral proteins, in contrast, are only transiently attached to the membrane.
Figure 1: Categories of membrane proteins. Integral membrane proteins and peripheral proteins are the two main types of membrane proteins. Integral membrane proteins can be further divided into polytopic, monotopic or lipid anchored protein. Peripheral membrane proteins transiently associate with the membrane itself or an integral membrane protein via non-covalent interactions.
Functions of membrane proteins
Membrane proteins play many essential roles in all living organisms. Below are a few examples of their diverse functions.
They can serve as receptors, which are proteins that can receive and transduce signals. Thus, proteins located in the plasma membrane can mediate communication between extracellular and intracellular environments by responding to signalling molecules, including hormones, neurotransmitters and more. For instance, G protein-coupled receptors (GPCRs) are a family of cell surface receptors with seven transmembrane helices that can bind to various external signalling molecules and trigger corresponding intracellular responses.
Intercellular joining and cell-cell recognition
Membrane proteins also play essential roles in cell adhesion, the process by which cells form contacts with each other, as well as attachment to the extracellular matrix. Additionally, some membrane protein-mediated interactions allow cells to distinguish between different types of neighbouring cells. The immunoglobulin superfamily proteins, for example, are involved in both intercellular joining and cell-cell recognition when facilitating an immune response.
In addition, some membrane proteins may contain active sites, which is the region of the enzyme that substrates bind. These proteins can catalyse specific chemical reactions and therefore said to possess enzymatic activities.
Figure 2: Schematic representation of various functions of membrane proteins.
Membrane proteins as transporters
In addition to the above roles, a large proportion of transmembrane proteins act as transporters. These are integral membrane proteins that facilitate the movement of ions and other molecules from one side of the membrane to the other. Biological membranes such as the plasma membrane act as a barrier that protects the cell from environmental toxins due to their selective permeability. Cells also have intracellular membranes that maintain different concentrations of proteins and small molecules in different compartments.
Transport proteins on the plasma membrane can regulate the transport of selective substances in and out of the cell. There are two main ways molecules can move across the membrane: facilitated diffusion where substances move down their concentration gradient; and active transport where energy from ATP hydrolysis is required to pump substances against their concentration gradient.
Depending on their mode of transport, transporter protein can be classified into channel proteins, which mediate facilitated diffusion; and carrier proteins, which mediate active transport.
Channel proteins open to both intracellular and extracellular environments simultaneously, providing an open pathway for specific ions or small polar molecules to simply diffuse down their concentration gradient across the lipid bilayer. The most common channel proteins are ion channels such as voltage-gated sodium channels and voltage-gated potassium channels, which selectively allow the passage of sodium and potassium ions, respectively. As these channels open and close in response to the electric potential across the cell membrane, they are important in shaping electrical signals such as action potentials in neurons. GLUT4, another example of a channel protein, is a glucose transporter found in skeletal and fat cells that allows uptake of glucose molecules from the blood.
Carrier proteins, in contrast, can only open to one side of the membrane at a time. They typically exploit the energy of ATP hydrolysis to induce this conformational change, collecting molecules from one side of the membrane and releasing them to the other side. The best-understood carrier protein is the sodium-potassium pump (Na+/K+-ATPase), which plays crucial roles in maintaining resting potential in the cell by pumping out three sodium ions and importing two potassium ions.
Figure 3: Two types of transporters. Channel protein mediates facilitated diffusion of ions and small polar molecules. Carrier protein mediates active transport of ions or molecules against their concentration gradient by changing their conformation.
The role of drug transporters in pharmacokinetics and drug-to-drug interactions
Drug transporters are expressed in a range of tissues including the intestine, kidney, liver, blood-brain barrier. As transporter proteins, they mediate the movement of compounds across the cell membrane. Therefore, they have a significant impact on the pharmacokinetics (what the body does to the drug), including the absorption, distribution, metabolism and excretion, of drugs.
The role of drug transporters is twofold. They can either enhance drug absorption by uptaking drugs into the cell or restrict drug absorption but promote disposition by pumping them out of the cell. They are particularly crucial to the distribution of drugs to tissues in the central nervous systems, which are protected by the blood-brain barrier and the blood-cerebrospinal barrier. Without access to the right drug transporters, it would be very difficult for drugs to enter the central nervous systems.
Drug transport proteins also modulate drug-drug interactions (DDI), which may arise when multiple drugs compete to bind to the same transporter. In other cases, co-administered drugs might affect the overall activity of membrane transport proteins, which in turn alters the pharmacokinetics of the drug of interest. As a result, clinically relevant DDI could reduce the efficacy of the drug or cause adverse drug reactions (ADRs).
Drug transporters have also been found to work cooperatively with drug-metabolizing enzymes (DMEs) during drug absorption, distribution and elimination. The suggested mechanism is that drug transporters recruit DMEs to the membrane, thereby helping them access the transported substrates. In this case, DME could catalyse phase I enzymatic reactions, which alters the pharmacological activity of the drug by changing its chemical structure. DMEs also catalyse phase II enzymatic reactions, which conjugates phase I metabolites (drugs that have been modified in phase I) with hydrophilic molecules, facilitating their excretion out of the body. Notably, the interplay between drug transporters and DMEs are highly dependent on the types of drug compounds and the drug doses.
Examples of membrane transporters with clinical relevance
Breast cancer resistance protein (BCRP)
Organic anion transporters (OAT1 and OAT3)
Organic cation transporter (OCT2)
Organic anion transporting polypeptides (OATP1B1 and OATP1B3)
Multidrug and toxin extrusion proteins (MATE)
The best-studied drug transporter in humans is p-glycoprotein, which is an ATP-dependent efflux pump that is responsible for exporting drugs out of the cell. This could facilitate drug elimination and restrict drug absorption.
P-glycoprotein is widely expressed in many tissues, such as the luminal membrane of the gut and blood-brain barrier. The expression level and activity of p-glycoprotein are tightly regulated, since this would influence the pharmacokinetics and drug-drug interactions, consequently affecting the efficacy and toxicity of drugs. Substrates of p-glycoprotein would activate this exporter, thus reducing the bioavailability of orally administered drugs as they are exported back into the gut lumen. Conversely, p-glycoprotein inhibitors would enhance the bioavailability of other drugs by promoting their absorption.
Furthermore, overexpression of p-glycoprotein is responsible for multidrug resistance (MDR) in chemotherapy. This is because p-glycoprotein has very broad substrate specificity, meaning that it can pump chemically diverse anticancer drugs out of the cell across the plasma membrane. Thus, the drug cannot reach the required concentration in tumour cells, preventing its action.
Breast cancer resistance proteins
BCRP is another ABC transporter that causes MDR in cancer. It is mainly expressed in the epithelia of the liver, gastrointestinal tract and placenta. Similar to p-glycoproteins, BCRP is also an exporter that pumps a wide range of therapeutic drugs out of the cell. This affects the pharmacokinetic properties of the drug by restricting intestinal absorption and promoting the excretion of xenobiotics.
Organic ion transporters
Some members of the SLC superfamily specialise in organic ion transport. OAT1 and OAT3 are two drug transporters of the SLC superfamily that mediate the transport of hydrophilic organic anions with low molecular mass, such as some uremic toxins, across the cell membrane. OCT2 is another protein in the SLC superfamily that mainly transport small, hydrophilic organic cations such as cisplatin, a drug used in chemotherapy. Unlike OATs and OCTs, which mediate the transportation of small organic molecules, OATPs mainly transport large hydrophobic organic anions such as statins, which are typically used to lower cholesterol levels. These organic anions and cation transporters are expressed in epithelial cells and distributed in almost every tissue in the body, thereby playing essential roles in drug absorption and disposition, as well as drug-drug interactions.
Multidrug and toxin extrusion proteins
Additionally, Multidrug and toxin extrusion proteins (MATE) are a newly emerging SLC transporter. They are exporters that can mediate the excretion of drugs and organic cations into urine and bile, therefore facilitating drug elimination.
Why should you care?
Membrane proteins play critical roles in the pharmacokinetics of many drugs, and therefore essential for the drug development process. Knowing the activity of transport proteins and the interactions between the transporters and the drugs are important for assessing the efficacy and safety of the candidate drug molecules. Besides, drug transporters could affect the bioavailability of administered drugs, which represents the proportion of drug dose that reaches the systemic circulation. Therefore, the study of membrane transporters provides crucial clinical indications on the optimal drug dose and administration routes when designing and developing new drugs.
However, membrane proteins are very difficult to study since it is hard to extract them out of the membrane. Once they leave the lipid environment, the flexibility and instability of membrane proteins also make structural and functional characterization difficult. These experimental challenges act as a major barrier to the drug development process. Since without an effective way to isolate membrane proteins, it would be hard to study the drug-transporter interactions and the effect of the transporter on drug distribution.
Researchers have developed some model membrane systems, such as liposomes, vesicular systems and planar bilayer. These model systems help the study of the structure and functions of membrane proteins, allowing a better understanding of their essential roles in pharmacokinetics and drug-drug interactions. All of this facilitates the development of new drugs for a wide range of diseases.
Helen Luojia Zhang
Imperial College London