- Transporters are membrane proteins that facilitate the transport.
- Present in all organisms, ~2000 genes in the human, ~7% of which are transporter related proteins.
- High passive permeability of a drug allows it to pass through easily and limiting the role of transporters in absorption. Low intrinsic permeability drug, in contrast needs help from transporters to pass through.
- Transporters expressed in liver and kidney along with metabolic enzymes, are key determinants of drug exposure/toxicity.
- Two major super-families are important for transport of drugs; ABC and SLC.
ABC transporters: efflux
- ABC transporters (ATP binding cassette)– Largest family of membrane bound proteins that mainly function as efflux transporters.
- Most ABC proteins are primary active transporters, which rely on ATP hydrolysis to actively pump the substrates across membranes.
- The structural architecture of ABC transporters consists minimally of two domains TMDs and two ABCs.
SLC transporters: drug uptake
- SLC transporters (solute carrier) are primarily involved in the uptake of small molecules into cells. Some currently marketed drugs, including diuretics, neuropsychiatric drugs and antidiabetic drugs are target for SLC transporters.
- SLC superfamily includes genes that encode facilitated transporters (rely on electrochemical gradient)and secondary active transporters(rely on ion gradients generated by ATP-dependent pumps transporting substrates against concentration gradient).
Roles of SLC transporters in human physiology
- Interestingly, the roles of many transporters in human physiology have been discovered through studies of Mendelian disease.
- Members of SLC families are required for intestinal absorption (from the gut lumen into the body) and renal reabsorption(from PCT into blood) of glucose and amino acids (needed for protein synthesis,)and for glucose uptake into neurons, erythrocytes, hepatocytes and other cell types.
- Metals often serve as essential cofactors for important enzymes, but toxicities may occur when they are present at excess concentrations. Zinc transporters of the SLC30 and SLC39 families and iron transporters of the SLC11 and SLC40 families regulate zinc and iron levels in the body, respectively.
- Similarly, water-soluble vitamins are essential for various processes but require transporters for cellular uptake; for instance, SLC19 family members transport folate and thiamine, SLC46 family members transport folate and the SLC52 family transports riboflavin.
- In the brain, neurotransmitters released into the synapse are taken back into presynaptic neurons through SLC1 and SLC6 transporters.
Broad Vs Narrow substrate specificities
- Transporters from both families, particularly those with broad substrate specificities such as :
- multidrug resistance protein 1 (MDR1; also known as P-glycoprotein or ABCB1)
- organic anion transporter 1 (OAT1; also known as SLC22A6) and those that serve in the absorption, distribution and elimination of structurally and pharmacologically diverse drugs.
- Such transporters may be the site of drug–drug interactions that underlie drug toxicities.
- Utilizing transporters as drug targets may require indirect methods, such as developing molecules that function as potentiators(activator) or correctors(inhibitors), or developing substrates that bypass the transporter.
- However, defects in functionally specific transporters with narrow substrate specificities (such as amino acid transporters in the SLC7 family) have been linked to many Mendelian diseases (or monogenic disorders).More than 80 SLC transporters have been implicated in monogenic disorders, indicating that this transporter superfamily could have substantial untapped therapeutic potential.
- Much remains unknown about SLC transporters — partly because many cell lines after multiple passages lose transporter expression and activity.
- However, knockout mice studies are useful to determine the relationship between transporters with similar transport functions.
- Slc-knockout mice have enabled the study of contributions of specific transporters when multiple transporters with overlapping substrate specificities are expressed in the same tissue.
- However, knockout of a transporter may be compensated by the availability or upregulation of other transporters that can transport the same substrate.
SLC transporters in disease
SLC transporters and Mendelian diseases
- Defects in a single transporter can result into a number of disease; some of these diseases are considered to be benign, others cause serious illness and death.
- Online Mendelian Inheritance in Man (OMIM) database.
SLC transporters and Common diseases
- SLC transporter gene polymorphisms that are associated with common diseases have generally been identified through the genotyping of candidate genes, or from GWASs.
- Genotyping – identifying small variations in genetic sequence(SNPs) by comparing an individual’s genotype (DNA sequence) with that of another sample or a reference sequence.
- Genome-wide Association Studies (GWASs)–to associate specific genetic variations with particular diseases. The method involves scanning the genomes from many different people and looking for genetic markers that can be used to predict the presence of a disease.
- The genome of a person is all DNA in an individual. The genotype is a specific region of DNA.
- Examples of transporter genes for which polymorphisms have been associated with human traits or disease include the following:
- Inflammatory bowel disease
- Skin colour
- Uric acid transporter(PCT)-A reduced-function variant of uric acid transporters will reabsorb less uric acid from the urine. As a result, hypouricaemia (lower level of uric acid in blood) can occur, thus reducing the risk of gout.
- Bilirubin transporter (Hepatic)– These transporters are responsible for uptake of conjugated bilirubin (product of haem catabolis into hepatocytes)from blood and then excretion of it into bile. Polymorphisms in the genes of hepatic transporters involved with bilirubin elimination pathway may lead to hyperbilirubinaemia and jaundice.
- SLC30A8,which encodes a pancreatic ß-cell-specific zinc transporter, a genetic polymorphism of diabetes which has been associated with diabetes.
- SLC14A1, which encodes a urea transporter,variants of which have been associated with bladder cancer.
- SLC4A7, which encodes a bicarbonate transporter, for which certain polymorphisms have been associated with , abnormal blood pressure or with breast cancer.
Strategies for targeting SLC transporters
Inhibition of transporter function
- High-throughput screening(HTS) of large compound libraries using cell lines that overexpress the transporter of interest can be used to discover lead inhibitor molecules.However, HTS also suffers from limitations:
- Assay interference due to aggregation of test compound can causes protein sequestration or nonspecific inhibition results in a false-positive hit.
- Fluorescent test compounds or particulate matter may interfere with the measurement of fluorescent substrate probe. A ‘pre-read’ control measurement after the addition of the test compound but before substrate addition can prevent this problem. Use of an orange or red fluorophore substrate probe can also help avoid this interference, as fluorescence and spurious light emissions from test compounds are more likely to occur at shorter wavelengths.
- Many SLC transporters function in both influx and efflux of their substrates, and net direction of flux is dependent on the substrate gradient or the gradient of the co- or counter-transported ions (in case of secondary/tertiary transporters).
- “Influx assays” are more frequently used in screening studies due to their simplicity. Cells grown on solid support are typically used for screening of inhibitors (drugs) that prevent the uptake/influx of a fluorescent substrate probe.
- For efflux ABC transporters, vesicular-transport assay is frequently used. Instead of adherent cells, membrane fractions that contain inside-out vesicles are prepared from cells that overexpress the efflux transporter. A fluorescent or radiolabelled substrate probe is added to each well in the absence (control) and presence of each test compound to determine its transporter-inhibiting activity.
- Examples of such efflux ABC transporters
- bile salt export pump (BSEP; also known as ABCB11),
- breast cancer resistance protein (BCRP; also known as ABCG2),
- canalicular multidrug resistance protein (cMRP; also known as ABCC2)
- Besides HTS, homology models of human orthologues can be used in molecular docking studies to design more-selective transporter inhibitors.
Enhancement of transporter function
Mechanism of transport
- ALTERNATING ACCESS MODEL
- Primary active transporters – require ATP to cause conformational change.
- For importers, the outward-facing conformation will have higher binding affinity for substrate.
- In contrast, the substrate binding affinity in exporters will be greater in inward-facing conformation.
- Importers ↑ in Outward facing conformation
- Exporters ↑ in Inward facing conformation
- ATP SWITCH MODEL
- Switching between the open and closed dimer conformations induces conformational changes in the TMD resulting in substrate translocation.
- ATP driven proton pumps or H+-ATPases are driven by the hydrolysis of ATP.
- P-type proton ATPase
- Single subunit P-type ATPase found in the plasma membrane.
- Humans have a gastric H+/K+ ATPase, primarily responsible for the acidification of the stomach contents.
- ER calcium pump is a P type antiporter(2H+/2Ca++).
- H+/K+ ATPase is a P type antiporter(3Na+/2Ca++).
- V-type proton ATPase
- Multi-subunit enzyme found in various different membranes where it serves to acidify intracellular organelles or the cell exterior.
- F-type proton ATPase
- Multi-subunit enzyme, also referred to as ATP synthase or FOF1 ATPase.
- It is found in the mitochondrial inner membrane where it functions as a proton transport-driven ATP synthase.
Transport of solute across biological membranes
- Basic mechanisms involved in the transport of solute across biological membranes are:
- passive transport (passive diffusion & facilitated diffusion)
- active transport (primary & secondary)
- PASSIVE DIFFUSION-It occurs down an electrochemical potential gradient of solute, it consists of 3 processes:
- partition from the aqueous to the lipid phase,
- diffusion across the lipid bilayer, and
- repartition into the aqueous phase on the opposite side.
- FACILITATED DIFFUSION– Diffusion of ions and organic compounds across the plasma membrane may be facilitated by a membrane transporter that does not require energy input (down their electrochemical potential gradient). Similar to passive diffusion, a steady state will be achieved when electrochemical potentials on both sides of the membrane become equal (equilibrative, not requiring energy).
- Active transport plays an important role in the uptake and and and efflux of drugs and other solutes. Depending on the driving force, active transport can be subdivided into primary and secondary active transport.
- Vesicle transport is a type of active transport that uses vesicles to move large molecules into or out of cells(Endocytosis, Exocytosis).
- PRIMARY ACTIVE TRANSPORT– directly couples with ATP hydrolysis.
- In mammalian cells, ABC transporter mediated unidirectional efflux of solutes across biological membranes.
- Na+/K+ ATPase pump
- SECONDARY ACTIVE TRANSPORT ( CO-TRANSPORT)-in contrast to primary active transport there is no direct coupling of ATP; instead,the electrochemical (ion) potential gradient (in contrast to chemical source of energy such as ATP) created by pumping ions out of the cell is used as energy source .The two main forms of this are:Antiport(Exchanger) and Symport(Co-transport).
- Antiport and symport processes are associated with secondary active transport(one of the two substances is transported against its concentration gradient, utilizing the energy derived from the transport of another ion (mostly Na+, K+ or H+ ions) down its concentration gradient.
- An ANTIPORTER carries two different molecules or ions, but in different directionsexample- Na+/K+antiporters(1 molecule of ATP is hydrolyzed as (3 Na+) are pumped out of the cell and (2K+) are pumped into the cell), Na+/H+antiporters, Na+\ Ca++ exchanger.
- A SYMPORTER carries two different molecules or ions, both in the same direction. Examples-Na+/Glucose symporter which cotransports one glucose molecule into the cell for every two Na+ ions.
- A uniporter carries one molecule or ion Example-glucose transporter (GLUT)). It may use either facilitated diffusion along diffusion gradient or against with an active transport process.
- Lipoproteins are complex particles composed of multiple proteins ( 80–100 proteins per particle ) and can transport up to hundreds of fat molecules per particle.
- Plasma lipoproteins are separated by
- hydrated density;
- electrophretic mobility;
- relative content of cholesterol, triglycerides, and protein
- Based on their density,five major classes of lipoproteins:
- very low-density lipoproteins (VLDL),
- intermediate density lipoproteins (IDL),
- low-density lipoproteins (LDL),
- high-density lipoproteins (HDL)
- With a size ranging from 5-17 nm, HDL is the smallest lipoprotein particles. Because of high cost of directly measuring HDL and LDL blood tests are commonly performed for the surrogate value, HDL-C, i.e. the cholesterol associated with ApoA-1/HDL particles.
Transporters involved in Pharmacokinetics
- Intestinal transporters-ABC transporters in the GI tract are located on apical and basolateral membranes.
- In the intestines, SLC transporters are used for both influx to the enterocyte and efflux into the blood (basolateral) or GI lumen (apical).
- Influx transporters expressed in intestinal brush-border improve drug absorption. Example: PEPT1, OATP-B, OATP-D & OATP-E, ASBT,. (PEPT1 mediates the transport of peptide-like drugs such as β-lactam antibiotics, ACEIs inhibitors and the dipeptide-like anticancer drug bestatin. )
- Efflux transporters, such as P-gp, MRP2, or BCRP, are also expressed on brush-border membrane of enterocytes and excrete their substrates into the lumen, resulting in limitation of net absorption. P-glycoprotein is a transporter protein that moves drugs out of cells and into the gut, urine, or bile.
- Active secretion of absorbed drugs is now becoming recognized as a significant factor in oral drug bioavailability. P-gp affects the absorption of many drugs because of its broad substrate specificity. Intestinal P-gp content correlates with the AUC after oral administration of digoxin, a P-gp substrate.
- High-fat meals may inhibit drug transporters, both influx and efflux.
- Fig: drug transporters expressed in enterocytes
- Hepatic transporters–ABC transporters in the liver are located on canincular and sinusoidal membranes.
- A liver sinusoid is a type of capillary, having discontinuous endothelium that serves as a location for mixing of the oxygen-rich blood from the hepatic artery and the nutrient-rich blood from the portal vein.
- The portal triad consists of the portal vein, hepatic artery, and bile ducts. Blood from portal vein and the hepatic artery flows toward the central vein. Bile produced by hepatocytes is collected into bile ducts via the bile canaliculi.
- Kupffer cells are located at the luminal side of sinusoids, while hepatic stellate cells (HSCs) are positioned in close proximity to hepatic sinusoidal endothelial cells (HSECs) at the “space of Disse”.
- Kupffer cells = phagocytic cells of liver sinusoids, involved in breakdown of RBCs),
- Hepatic stellate cells (HSCs) = mesenchymal cells playing vital roles in liver physiology and fibrogenesis.
- “Space of Disse”, a location between hepatocytes and a sinusoid.
- The canal of Herring = junctional region between hepatocytes and bile ducts.
- Canalicular membrane exports molecules into the bile using both the ABC and SLC transporters.
- Sinusoidal membrane exports molecules to the blood using the ABC transporter.
- Sinusoidal membrane imports molecules to the hepatocytes (liver cells) using the SLC transporter. In the liver, drugs enter through the sinusoidal membrane using SLC transporter.
- Fig: Hepatic transporters.
- Fig: Liver Lobule
- The clearance rate for GFR is 120 ml/min.
- Requirements for glomerular filtration are:
- – small molecular weight (MW<400)
- – non-ionized
- – non-protein bound
- The clearance for active tubular secretion is 425 to 650 mL/min
- Requirements for active tubular secretion:
- – week acids and bases
- – dependent on RPF (Renal plasme flow~Renal blood flow; roughly 25% of cardiac output, amounting to 1.2 – 1.3 L/min in a 70-kg adult male.)
- – requires energy input
- – carrier mediated, capacity-limited (saturable
- The clearance for tubular reabsorption<120 mL/min
- Requirements for tubular reabsorption:
- – weak acids or bases dependent on pH of fluid in renal tubule and pKa of drug.
- -passive or active
- – non-ionized
- Renal transporters-Transporters in the kidneys are predominantly located in PCT (proximal convoluted tubule).
- ABC transporters in the kidneys are located on apical membrane leading to lumen (urine).
- SLC transporters in the kidenys are located on basolateral (blood, influx to the proximal tubule cell) AND apical (efflux to the lumen)membrane.
- Apical membrane exports molecules to the GI lumen (bile) or tubule lumen (kidneys).
- Fig: drug transporters expressed in kidney
- Blood-brain barrier
- Blood-testes barrier
- efflux transporters limit drug penetration (eg. P – glycoprotein in BBB)
- Fig: drug transporters expressed in brain
TRANSPORTER and PHARMACODYNAMICS
- TRANSPORTERS AS DRUG TARGETS
- TRANSPORTER-MEDIATED ADVERSE DRUG RESPONSES
- TRANSPORTERS IN DRUG RESISTANCE
TRANSPORTERS AS DRUG TARGETS
- Transporters in reuptake of neurotransmitters (SLC6 family-Na+/Cl–-dependent transporters )
- Cholesterol transporters in cardiovascular disease
- Na+/H+antiporters in hypertension
- Glucose transporters in metabolic syndromes
Transporters involved in neuronal reuptake of neurotransmitters
- Transporters are also involved in neuronal reuptake of neurotransmitters in the presynaptic neuron and regulation of their levels in the synaptic cleft.
- They belong to two major superfamilies, SLC1 and SLC6.
- These transporters may serve as pharmacologic targets for neuropsychiatric drugs.
- Transporters in both families play roles in reuptake of GABA (γ-aminobutyric acid), glutamate, and the monoamine neurotransmitters NA, 5-HT(5-hydroxytryptamine or serotonin), and dopamine receptors.
Cholesterol transporters in cardiovascular disease
- Reverse cholesterol transport (RCT) is a mechanism by which HDL-c removes excess cholesterol from peripheral tissues and delivers them to the liver, where it will be redistributed to other tissues or excreted in bile.
- In contrast low density lipoprotein (LDL) transports cholesterol from liver to peripheral tissues including adrenal glands and gonads. Cells use cholesterol but too much can build up in arteries(atherosclerosis).
- Moderate physical activity can help raise high-density lipoprotein (HDL) cholesterol, the “good” cholesterol.
Na+/H+antiporters in hypertension
- Na+/H+antiporters play a major role in pH and Na+homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants.
- Angiotensin II upregulates Na+/H+antiporters in PCT (proximal convoluted tubule) to promote Na+ reabsorption and H+ secretion.
- Angiotensin II, a peptide hormone of renin–angiotensin-aldosterone system regulates blood pressure (by causeing vasoconstriction ). It also stimulates the release of aldosterone from the adrenal cortex to promote sodium retention by the kidneys.
- Angiotensin I is converted to angiotensin II through removal of two C-terminal residues by the enzyme angiotensin-converting enzyme (ACE), primarily in lungs but also in endothelial cells, kidney epithelial cells, brain)
Glucose transporters in metabolic syndromes
- There are two main types of glucose transporters, which can be divided into many more subclasses.
- Sodium–glucose linked transporters or SGLTs (secondary active)
- Facilitated diffusion glucose transporters or GLUT (passive)
- GLUT1 is insulin-independent and is widely distributed in all cells including muscles.
- Intramuscular glucose is used for RNA synthesis (pentose phosphate pathway) , glycoproteins synthesis (hexosamine pathway),and for transcriptional control of certain genes. These metabolic reactions need a continuous fuel(glucose) flux independent of nutritional status and insulin secretion.
- Glutamine amino acid is also synthesized from glucose in the skeletal muscle. Continuous maintenance of sufficient glutamine concentration in blood is necessary for the proper functioning of the immune system and survival.
- GLUT4 is insulin-dependent transporter which is responsible for the majority of glucose transport into muscle and adipose cells in anabolic conditions (to facilitate storage of glucose in muscle and adipose tissue in insulin-stimulated conditions, such as after a meal).
- GLUT1 seems to be coupled with hexokinase I, and GLUT4 coupled to hexokinase II.
TRANSPORTER-MEDIATED ADVERSE DRUG RESPONSES
- Transporter-mediated adverse drug responses can be classified into 3 categories:
- Increased drug concentrations in toxicological target organs(e.g., liver, kidney and brain ) due to enhanced uptake or reduced efflux of drug.
- Increased plasma concentrations of drug due to decrease in the uptake and/or secretion in clearance organs (e.g., liver and kidney).
- Increased plasma concentration of an endogenous compound (e.g., a bile acid) due to a drug’s inhibiting the influx/efflux of endogenous compound in its eliminating or target organ.
TRANSPORTERS IN DRUG RESISTANCE
- Membrane transporters play crucial role in the development of resistance to several drugs like-anticancer drugs, antimicrobials and anticonvulsants.
- Reduced expression of influx transporters needed for uptake of some anticancer drugs ( & access to tumor) such as folate antagonists, nucleoside analogs(cytarabine, gemcitabine, mercaptopurine, 5-FU), and platinum complexes.
- Enhanced expression of efflux transporters of hydrophobic drugs. For example,
- ABCB family– MDR(Multidrug resistance). P-glycoprotein (P-gp) (MDR1/ABCB1) is overexpressed in tumor cells after exposure to anticancer agents to pumps out the anticancer drugs.
- ABCC family- MRP (Multidrug Resistance-associated Protien) or CFTR(Cystic Fibrosis Transmembrane conductance Regulator). Over-expression of MRP4 (multidrug resistance protein 4) is associated with resistance to antivirals and anticancer drugs.
- ABCG2 or BCRP (human breast cancer resistance protein) restricts the distribution of its substrates (drugs) into organs (brain, testes, placenta, and across the gastrointestinal tract (GIT) and also mediates both biliary and renal excretion, and occasionally direct gut secretion.
- MATE familty (Multidrug and toxin extrusion protein)–they are SLC efflux transporters that have affinity for organic cations. Example- H+/drug antiporters: imports H+ into the cell in exchange of a drug molecule (exported out of the cell).
TRANSPORTERS VERSUS CHANNELS
- Both channels and transporters facilitate the membrane permeation of inorganic ions and organic compounds.
- Water crosses the membranes through channels “Aquaporins”.
- Channels do have 2 stochastic (randomly determined) primary states/conformations-open and closed. Only in the open state the channels act as pores for the selected ions and then return to the closed state as a function of time.
- Channels can be regulated by several mechanisms: volatge gated, ligand- gated or mechanical-gated.
- Channel structure determines its selectivity.
- Sodium channels have key role in generating membrane potential.
- A transporter forms an intermediate complex with the substrate (solute), and subsequent conformational change in the transporter induces translocation of the substrates to the other side of the membrane.
- Transporter-mediated membrane transport is characterized by saturability and inhibition by substrate analogs. The flux of a substrate (rate of transport) across a biological membrane via transporter-mediated processes is given by the Michaelis-Menten equation. The turnover rate constants of typical channels are 10^6 to 10^8 s–1; those of transporters are, at most, 10 to 1000 s–1.
where Vmax is the maximum transport rate and is proportional to the density of transporters on the plasma
membrane, and Km is the Michaelis constant, which represents the substrate concentration at which the flux
is half the Vmax value. Km is an approximation of the dissociation constant of the substrate from the
- Chapter 5: Membrane Transporters and Drug Response. Goodman and Gilman’s Manual of Pharmacology and Therapeutics, 2e.