Drug transporters Flashcards
What are membrane transporters roles?
- Regulate distribution and biovalailability of drugs
-Remove toxic metabolites and xenobiotics
-Protect haematopoietic stem cells from toxins
What does cellular membranes do to solutes?
Partition solutes to generate gradients through membrane transporters.
What is the function of membrane transporters?
Carry solutes across cell membranes which otherwise be impermeable to them
* Exploit gradients for various purposes (energy generation for example)
Mention an example of membrane transporters
F-type ATPase
Why are membrane transporters important?
Transporters play an important role in absorption, distribution and excretion, and as such are important in:
* Pharmacokinetics
* Pharmacodynamics
* Drug-drug interactions
* Toxicity
* Drug delivery
* Personalised healthcare (precision medicine)
Mention characteristics of transporter-targeting drugs
- Currently all inhibitors
- Usually derived initially from natural substrate
- Suitable chemical modification to resist metabolism, maybe with ‘bulky’ substituents
How remove of toxic metabolites works?
-from the cells into urine, bile and the intestinal lumen
– out of the brain across the blood brain barrier
What do transporters influence in drug disposition?
- Drug action
- Drug metabolism
- Drug resistance
*
Mention the three major families of transporters
- P-type ATPases
- ATP – Binding Cassette (ABC) transporters
- SoLute Carrier (SLC) family members (2nd largest family)
Describe P-type ATPases
- Ion pumps that use energy from ATP hydrolysis
- Ubiquitous membrane proteins
41 members, often heterodimeric (a and b) - Autophosphorylation of catalytic asp
Hence P-type - Six subfamilies
Five transport inorganic cations (probably) - Can be found on cell-surface and intracellular organelles
Describe ABC transporters
- Ubiquitous membrane proteins
48 in seven families A-G - Found in cell-surface and organellar
- Utilise ATP hydrolysis to facilitate solute transport out of the cytosol
- Mostly monomeric
Individual subunits typically made up of two groups of nucleotide binding 6TM-spanning domains
Mention examples of multi-drug resistance drugs
- ABCB1 (MDR1, pgp)
- ABCC1 (MRP1)
- ABCG2 (ABCP)
What is the function of multi-drug resistance drugs
Responsible for the efflux of a huge variety of exogenous compounds, notably chemotherapeutic agents
* reduces absorption at the apical membrane of epithelial cells
Depends of how the distribution of drugs across the membranes is needed we can define three types of transporters. Mention it the three types binding.
1.Pump
2.Carriers
3. Channels
Mention characteristics of ABC transporter structure
- Mostly 12 TM domains, divided 6 and 6 (TMD1 and TMD2).
- Nucleotide binding domain (liker between the two TMD1 and TMD2) there is where ATP binds. Generally exist as dimers.
- N terminus is in cytoplasm and C terminus too.
- Some could be only 6 TM domains, Some of them may be 18 TM domains.
Mention the 9 p-type ATPase subfamiles
- Phospholipid
- SERCA
- SPCA
4.Na/K
5.H/K - PMCA
- Mn?
- Cu
9.b subunits
Why are ABC transporters, active transporters?
They require energy in the form of ATP to
translocate substrates across cell membranes
How do ABC transporters work along with ATP to transport molecules
These proteins harness the energy of ATP binding &/or hydrolysis to drive conformational changes in the TMD and consequently transport molecules
Where is ABCB1 - P-glycoprotein expressed?
Expressed at brush border
membranes of enterocytes
What is the function of ABCB1 - P-glycoprotein
Functions as an efflux pump for
xenobiotics before they can access the
portal circulation
How do ABCB1 - P-glycoprotein affect cancer patients?
It is a frequent cause of treatment
failure in cancer patients
Mention examples of xenobiotic substrates of ABCG2 (BCRP) transporter?
- Apigenin
Dietary flavonoid - 5-Fluorouracil
Anti-cancer - Mitoxantrone
Anti-cancer - Tacrolimus
Immunosuppressant
Mention examples of inhibitors of ABCG2 (BCRP) transporter?
- Gefitinib - EGFR inhibitor
Reverses drug resistance, increases cellular
drug accumulation inhibiting ABCG2-mediated drug efflux - Febuxostat - XO inhibitor
Treating gout
Which are the functions of ABC transporters?. Give an example of each function
1, Metabolic funtions
- Cholesterol export (ABCG2)
– Fatty acid metabolism (ABCD)
2. Signalling functions
- Prostaglandin, leukotriene (ABCC1) export
3. Ion movements
Cl-, HCO3
(CFTR, cystic fibrosis transmembrane
regulator) (important specific for this family due to is link to the disease cystic fibrosis which affects lungs)
Describe SLC transporters
- Ubiquitous membrane proteins
- Second largest family of membrane proteins
– ~430 members in 65 families - Present on cell-surface and organellar
- Limited sequence/structural homology
– 6TM-13TM - Multiple orphans (~20 %)
What are the metabolic functions of SLC transporters
Amino acids (~25 %), fatty acids, peptides, sugars, nucleosides, bile acids, urea, vitamins, nucleotides (mitochondria)
Mention the best recognised ABC family members
- P-glycoprotein, P-gp – encoded by ABCB1, also known as MDR1
- CFTR : Cystic fibrosis transmembrane regulator
What are the signalling functions of SLC transporters?
Mention examples too
- Amino acid transmitters (glutamate, glycine, GABA)
- Amines (noradrenaline, adrenaline, 5HT, dopamine, ACh)
What are some of the ions that can be moved by SLC
Cu, Zn, Mg, Fe, PO4 3-, SO4 2- accumulation
Mention the two distinct domain for all ABC transporters
- The transmembrane (TM) domains.
- The nucleotide binding domain/s (NBD).
Mention the two distinct domain for all ABC transporters
- The transmembrane (TM) domains.
- The nucleotide binding domain/s (NBD).
Why there is complex pharmacology involved in ABC transporters?
They have multiple overlapping binding sites
Which is the biggest type of ABC transporters?
ABCA with 1200 to 2600 aas
What are the characteristics for the transmembrane (TM) domains of ABC transporters?
- a helices embedded in the membrane bilayer
- Recognise a variety of substrates
- Undergo conformational changes to transport the
substrate across the membrane - Variable sequence and architecture of TMDs reflects the chemical diversity of substrates that can be translocated
What are the characteristics for the nucleotide binding (NBD) transporters?
Its located in the cytoplasm and Highly conserved sequences
What type of compounds Influx ABC trasnporters transport?
Nutrients such as
-Maltaso
-Histidine
-Arabinose
-Galactosa
How are SLC transporters organized into families?
Based on 20 to 25% similarity
What is the usual nomenclature for the SLC transporters?
SLCnXm
* Where:
– SLC = root name, SoLute Carrier
– n = integer representing family (0-52)
– X = single letter denoting subfamily (A, B, C…)
– m = integer representing individual family member (isoform)
What does antiport or counter-transport means?
That two different molecules or ions are transported at the same time but in opposite directions
* One species is allowed to flow from high concentration to a
lower concentration (often Na+) while the other species is
transported simultaneously to the other side
Explain the ABC transporters mechanism of
transport (ATP-Switch model)
There are 2 principal NBDs conformations:
1. Formation of a closed dimer upon binding two ATP molecules
2. Dissociation to an open dimer facilitated by ATP hydrolysis and release of
inorganic phosphate (Pi) and adenosine diphosphate (ADP)
Mention an example of Na+/Ca2+ counter-transporters and how do they work
(NCX1-3/SLC8A1-3)
* Na+ binds to the transport carrier protein on its exterior side,
and Ca2+ bound to the same protein on the membranes interior
side
* Once both are bound, a conformational change occurs which
releases energy and the sodium ion is transported to the
interior and calcium to the exterior
What induce the conformational changes in the TMD ? What is the result of this conformational changes?
Switching between the open and closed dimer conformations induces
conformational changes in the TMD resulting in substrate translocation.
Where are Na+/Ca2+ counter-transporters found?
This transporter is situated on almost all cell membranes
Where ABCG2 are expressed?
- High expression in the brain
2.Expression at apical membrane of renal & intestinal epithelial cells
- Localized to canalicular membrane of hepatocytes
Mention the functions of ABCG2
Consist on excretion role
1. Restricts intestinal absorption of pharmaceuticals.
2. If its present in brain, placenta- Defence of organs against xenobiotics
3. Protects haematopoietic stem cells against haem-induced toxicity
4. Exports nutrients into milk
5. Exports urate from kidney
6. Exports chemotherapeutic drugs from cancer cell
Mention examples of symporters
- Na+/amino acid co-transport
– SLC1A glu/asp (EAAT) ala/ser/cys (ASCT)
– SLC6A gly/pro/leu/met/iso/val/asn/phe/ala/ser/thr
– Widely expressed, incl. gut epithelium - H+/amino acid co-transport
– SLC15A di- and tri-peptides (PEPTs)
– SLC36A (PATs)
– Gut and kidney epithelia
Where are Solute Carrier Organic Anion (SLCO) transporters expressed?
In the liver: SLCO1B1, 1B3, 2B1 mainly located in sinusoidal membrane of hepatocytes
Which are the potential function of solute carrier (SLC) transporters?
SLC are potential to function as:
- Antiporters (exchanger)
– Symporters (co-transporters)
– Equilibrative transporters
– One family (SLC27) has enzymatic function
– Some are electrogenic - translocation of net charge across the
membrane (SLC1A7, cf. ion channels)
What suggest that SLCs lack an ATP binding domain?
SLCs lack an ATP binding domain, so exploit ion gradients to facilitate solute transport.
A polymorphism in which gene is associated with impaired transporter function?
SLCO1B1 gene
Mention examples of SLCO1B1/OATP1B1 endogenous substrates
Cholic acid
PGE2
Thyroxine
17b-Estradiol glucuronide
Name the three types of transport show in the image (export and import directions)
Mention examples of SLCO1B1/OATP1B1 xenobiotic substrates
- Rifampicin
Antihypertensive - Atorvastatin
Antihypercholesterolemic - Bosentan
Antihypertensive - Enalapril
Antihypertensive
Explain the Neurotransmitter Transporters mechanism.
Where is Solute Carrier Organic Anion (SLCO) SLCO1B3/OATP1B3 majorly expressed?
- In the liver
- Sodium-independent uptake of nonpolar drugs and hormones from the portal vein
Which types of solutes SLC transport?
-Charged and uncharged organic molecules
– Inorganic molecules
– Gas ammonia
What is the importance of SLCO1B3/OATP1B3
A critical role in bile acid and bilirubin
transport
– Mutations in this gene lead to Rotor syndrome hyperbilirubinemia (mild, rare)
How SLC as a facilitative transporters work?
- solutes flow down electrochemical gradients
Describe the SLC22 family and where are they expressed
The SLC22 family of transporters is mostly composed of non-selective transporters, which are expressed highly in liver, kidney and intestine
How SLC as a How SLC as a – Secondary active transporter work?
Solutes flow uphill against
electrochemical gradient by coupling to transport of second solute that flows down gradient, overall free energy change is still favourable
Describe the membrane topology of SLC22
Membrane topology is predicted to contain 12 TM domains with intracellular termini, and an extended extracellular loop at TM 1/2
Why are SLC22 important?
How is SLC22 family divided?
The family may be divided into three subfamilies based on the nature of the substrate transported:
– organic cations (OCTs),
– organic anions (OATs) and
– organic zwiterrion/cations (OCTN)
How Secondary active transport: symporters work?
Symport or co-transport means that a molecule is allowed to be transported from high to low concentration while
“pulling” another molecule with it from low to high
concentration
Where Secondary active transport: symporters occurs?
These transporters occur especially through the epithelial cells of the intestinal tract and the renal tubules of the kidneys to enable absorption of substances into blood.
Mention examples of SLC22A6/OAT1 xenobiotic substrates
- p-Aminohippuric acid
Diagnostic aid - Azidothymidine
Antiretroviral - Simvastatin
Antihypercholesterolemic - Telmisartan
Antihypertensive
Mention examples of SLC22A6/OAT1 inhibitor
Probenecid
Anti-gout
High potency, but non-selective
Give an example of Secondary active transport: symporters.
Na+/glucose co-transporter (SGLT/SLC5A)
– On its exterior side the transport protein has 2 binding sites, one
for Na+ and one for glucose.
– When both are bound to the protein, there is a conformational
change allowing the electrochemical gradient to provide the energy needed to transport both of these molecules into the cell
Mention examples of SLC22A2/OCT2 xenobiotic substrates
- Amiloride
Diuretic - Metformin
Antidiabetic - Procainamide
Local anaesthetic - Ranitidine
Anti-ulcer - Varenicline
Anti-craving
Mention examples of SLC22A2/OCT2 inhibitor
Decynium-22
Mention 3 SLC families transporters
- OATP - Organic Anion Transporting Polypeptides
- OAT - Organic Anion Transporters
- OCT - Organic Cation Transporters
Where are mainly transporters expressed?
Many transporters expressed ubiquitously
Some demonstrate higher/selective expression in certain regions
(i.e liver, kidney)
In which part of the small intestine are mostly transporters expressed?
Multiple transporters are expressed in the brush-border membranes of intestinal epithelial cells
What is the purpose of transporters in the small intestine?
They are involved in the efficient absorption of nutrients or endogenous compounds
Mention examples of influx transporters that are involved in improving absorption in the GI tract
PEPT1, OATP, ASBT
Which transporter mediates the transport of peptide-like drugs?
Mention examples
PEPT1
* b-lactam antibiotics
* ACE inhibitors
* The dipeptide-like anticancer drug bestatin
What is Pomaglumetad methionil
Pomaglumetad methionil – prodrug that is rapidly
absorbed and hydrolysed
Where is OATP transporter present ? what are the some of the functions?
OATP present on the BBB,promote clearance through metabolism, transport a number of anti-cancer drugs.
Give an example of OATP transporter inhibitor
Tyrosine kinase inhibitors reported to inhibit OATP (potential for DDIs!)
– Pazopanib and nilotinib have inhibitory potential towards OATP1B1
– Vandetanib has inhibitory activity towards OATP1B3
What is OATP polymorphism associated?
OATP polymorphism is associated with the incidence of simvastatin myopathy.
Which is the relation between OATP polymorphism and the inward sodium gradient?
SLC22/OAT are indirectly dependent on the inward sodium gradient, which
is a driving force for re-entry of dicarboxylates into the cytosol
Where are OATPs expressed?
Through the body but they play a critical role in the liver
Why are OATPs important?
Play critical roles in the absorption, distribution and excretion of xenobiotics
Which OATPs are selectively expressed in the liver? What is its function?
OATP1B1 and 1B3 are selectively expressed in the liver, localized to the basolateral membrane of hepatocytes
* Facilitate access to intracellular targets/metabolic
enzymes
Where are OATs expressed?
Throughout the body (kidneys important)
Transporters demonstrate differential expression across the GI tract. Which is the major percentaje contribution transporter in the small intestine and colon?
Small intestine PEPT1 50%
Colon ABCC3 36%
Which OATs are highly expressed in the kidney?
– OAT1 and OAT3 are highly expressed in the kidney, at the basolateral membrane of proximal tubules
– OAT4 expressed in kidney, at the apical membrane of proximal tubules
Why are OATs important?
They have a role in drug excretion
Where are ABCG2 expressed?
- Expression is widespread
- Presence on the luminal side of brain capillaries suggests a brain-to-blood transport direction
What is the inducer of ABCG2?
PPARalpha
Where are SLC9 Na+/H+ exchange (NHE) family expressed?
Transporters in liver & kidney
What is the function of SLC9 Na+/H+ exchange (NHE) family Apical membrane of PCT cell
- Transports 1 Na+ in and 1 H+ out
Antiporter - Uses gradient established by Na+/K+ ATPase
- Target of amiloride
- Essential in maintaining blood pH at 7.4
Where are this transporters ABCB1/P-gp and what is its function?
In the brain / drug exclusion
Provided the role of Transporters in intestinal epithelium. Give an example
They are involved in the efficient absorption of nutrients or endogenous compounds
PEPT1 mediates the transport of peptide-like
drugs
– b-lactam antibiotics
– ACE inhibitors
– The dipeptide-like anticancer drug bestatin
Which drugs may be affected by ABCB1/P-gp?
Anti-histamines (hH1R antagonists)
Hydroxyzine and Cetirizine
Which tranporters are expressed in the brush-border membranes of intestinal epithelial cells?
-OATP
-PEPT1
-ASBT
-MCT1
Provided an example of transporter in theintestinal epithelial for drug accumulation?
SLC15A1/PEPT1 and SLC36A1/PAT1
- Exploit H+ gradient
- Transport a range of substances, including:
– Di- and tripeptides (protein metabolites)
– 5-aminolevulinic acid
– Bacterial peptides (fMLP)
Which is the relation between P-gp and absorption?
P-gp affects the absorption of many drugs because of its broad substrate specificity
What are the role of efflux transporters in intestinal epithelium? Give 3 examples
The efflux transporters excrete substances into the lumen of the intestine, thus limiting net absorption.
eg.P-gp, MRP2, BCRP
Which is the portion of solutes reabsorbed in the proximal convoluted
tubule in the kidney? Name the slutes reabsorbed
60 % of all solute
- Glucose and amino acids
* Bicarbonate
* Inorganic phosphate and water
Give 3 transporters in liver & kidneyc vvc vc
Give 3 transporters in liver & kidneyc vvc vc
What are the OCTs transporters? Give 2 examples and where are those located?
Are multi-specific uptake transporters expressed in numerous epithelia
throughout the body.
OCT1
* Highly expressed in the liver, at basolateral
membrane of hepatocytes
– OCT2
* Highly expressed in kidney, at basolateral
membrane of distal convoluted tubules
Explain the process of Na+/K+ ATPase transporter in the liver & kidney?
Primary active transporter.
Transports 3 Na+ out of cell (into interstitium)
and 2 K+
into cell whilst hydrolysing one
molecule of ATP
* Reabsorption of filtered Na+
* Generates/maintains electrochemical gradients
* Gradients used by other transporters
Why is it important to understand drug-metabolizing enzymes ?
It allows the prediction of DDIs in vivo from in vitro data.
What is the purpose of International Transporter Consortium (ITC) ?
- To determine if a similar predictive perspective exists for drug transporter proteins
- Comprises transporter experts from industry, academia and regulatory agencies
What are some of the things that the FDA provides for New Drug Applications (NDA) and Biologic License applications (BLA)?
Provides recommendations for with in vitro and in vivo studies of drug metabolism, drug transport, and drug-drug or drug-therapeutic protein interactions
Focus on drug transport
Indicate each process of the following image about drug transporters
What has been documented in recent years by the FDA regarding transporters?
- All investigational drugs should be evaluated in vitro to determine whether they are a potential substrate of P-gp or BCRP
- Investigational drugs should be evaluated in vitro to determine whether they are a substrate of hepatic uptake transporters OATP1B1 or OATP1B3, when their hepatic pathway is significant
- Similarly, investigational drugs should be evaluated in vitro to determine whether they are a substrate of OAT1 or OAT3 or OCT2 when renal active secretion is important
The need for further in vivo drug interaction studies will be based on in vitro evaluation
How you can identify key transporters as relevant targets for DDI?
– Decision making flowcharts to identify substrates/inhibitors of transporters
– Recommendations on substrates and inhibitors
– Directions for essential in vivo DDI studies for transporters
– Unbound concentration of drug is more relevant for DDI predictions
– Use of validated in vitro transporter model
What needs to be considered for therapeutic proteins according to FDA?
The potential for interactions with drug products
What should include the development of a drug based on the FDA Guidance 2017?
– Identification of the principal routes of elimination
– Quantitation of the contribution by enzymes and
transporters to drug disposition
– Characterization of the mechanism of drug-drug
interactions
– in vitro drug transporter studies with and without
inhibitor or with and without expression of the
transporter
What study needs to be conducted for cytokine and cytokine modulator investigational drugs?
Studies should be conducted to determine the TP’s effects on CYP enzymes or transporters.
How can in vivo evaluations be done for therapeutic proteins?
The in vivo evaluations of TPs in targeted patient populations can be conducted with individual substrates for specific CYP enzymes and transporters, or studies can be conducted using a “cocktail approach”
What are some common cell-base assays?
- Uptake inhibition assays
- Bidirectional and unidirectional transport assays
- Cytotoxicity assays
These assays utilize fully differentiated monolayers of mammalian cell lines to conduct a range of studies
Which are the two different mechanisms where drug transporters may be involved in organ toxicity?
- Disruption of the transport function of controlling the influx and efflux of
essential nutrients and ions - Transporter regulation allowing intracellular drug concentrations to reach
unsafe levels leading to organ- specific injuries
What are some common membrane permeability and transport assays?
For transport assays and membrane permeability functions, cells are seeded onto semipermeable membrane support that develops into a monolayer resembling the physiological epithelial barrier in vivo
* Caco-2, Calu-3
Primary human epithelial cell lines
What are the effects of altered transporter kinetics due to inhibition, saturation or genetic polymorphisms?
Altered transporter kinetics due to inhibition, saturation or genetic polymorphisms may significantly alter the systemic or organ level of drug exposure, and subsequently result in altered PK, PD and safety profiles.
