ABC transporters and multidrug resistance

2 main transporter families

 ATP-binding cassette (ABC) superfamily, depend on ATP binding

 Solute carrier (SLC) superfamily

-OAT – organic anion transporter

-OATP – organic anion transporting polypeptide

-OCT – organic cation transporter

-MATE – multidrug and toxin extrusion protein

what are ABC transporters

-membrane proteins that couple substrate transport to ATP hydrolysis

-these proteins can be importers or exporters of substrates (not both)

-exporters/efflux transporters: ABCB1/MDR1/P-gp (mammals)

-importers/influx transporters: found in prokaryotes e.g. maltose or methionine uptake transporter in E. coli

-no transport function but associated with ion channels: CTFR, SUR1

-typically they are expressed in cells with excretory or barrier functions: liver, intestine, kidney, BBB, blood placenta and testis barrier

-mostly expressed in a polarised manner and individual ABC transporters are expressed on either apical side or basolateral side (not both)

-typically protect the body against xenobiotics

-some have a normal physiological role (e.g. bile acid transport in liver or regulation of insulin release in pancreas)

structure of ABC transporters

-48 human ABC transporter genes

-7 subfamilies: a-g

-4 domains: 2 nucleotide binding domains (NBD) which bind and hydrolyse ATP; 2 transmembrane domains (TMD) which bind and transport substrates

-NBDs are highly conserved

-TMDs are less conserved

-a full transporter as 2 transmembrane domains that form the channel and the 2 ATP binding domains

-across the subfamilies there are also half-transporters, encode for one transmembrane domain, and 1 ATP-binding domain; they must form homo or heterodimers to function

-some members have very narrow which others have wide substrate specificity

transporter protein in the intestine

-to circulate, drugs need to enter enterocytes through brush border membrane and the cross the basolateral membrane into hepatic portal vein

-the ABC transporters interfere with this process and pump compounds such as drugs back into the lumen

transporter proteins in the liver

-drugs often need to be transported into hepatocyte from circulation across sinusoidal membrane

-drugs (usually after metabolism) need to be transported out either across canalicular membrane (to bile) or sinusoidal membrane (back to blood for renal excretion)

transport proteins in the BBB

-transporters at BBB prevent many potentially toxic compounds reaching the brain

-KO mice studies show drug accumulation in the brain and toxicity, shown by increased sensitivity to xenobiotics etc

multidrug resistance

-defined as a phenomenon where cells/cancer cells become resistant to a broad range of chemotherapeutics/drugs

-poses difficult clinical issues

-often its not the primary tumour that kills the patient

-resistance can be intrinsic or acquired

e.g. decreased uptake by cell, increased metabolism of drug in cell, alteration in drug target, enhanced drug efflux

-enhanced drug efflux is mainly down to high expression of ABC transporters

-MDR1, MRP1, ABCG2, main transporters of interest

P-glycoprotein (MDR1)

-encoded by ABCB1 gene

-1976, Victor Ling showed that the resistant cells have high levels of protein called P-glycoprotein on cell surface

-shown to be present in normal tissues and many tumour cell lines

-now known that human ABCB1 gene on chromosome 7 codes for this protein

-cells had varying permeability to the drugs (hence the P)

-has 2 transmembrane domains that vary along members

-each domain has 6 transmembrane alpha helices

-inbetween the transmembrane domains there are 2 ATP binding domains

-normally would sit open inward, towards the cytoplasm, allowing the substrate to enter and bind

-then ATP binds and is hydrolysed that eventually leads to an efflux of your substrate

-ATP binding causes the coming together of the NBDs

role of PGP

-removal of xenobiotics from cells

-pumps out xenobiotics from enterocytes following initial absorption

-transports xenobiotics into bile across canalicular membrane

-prevents access of many xenobiotics to brain

-transports xenobiotics into lumen of kidney on brush-border membrane

PGP KO studies

-in mice

-they kept dying, but discovered that they were using ivermectin as an anti-lice treatment

-this is an antibiotic, but the PGP was knocked out, so it was getting into the brain

-showed increased absorption and decreased excretion of a number of drugs

border collies

-have a 4 base pair deletion within their ABCB1 gene

-premature stop, so no expression of pgp

-about 75% of pure bred collies don’t have a functional pgp

-interacts with various agents such as anaesthesia

pgp and CYP2A4

-substrate specificity of Pgp overlaps with CYP3A4 (important in drug detoxification)

-Pgp is also induced through PXR receptor by compounds such as rifampicin

-St John’s Wort is a herbal remedy for low mood and mild anxiety, increases P-gp expression and hence efflux activity, induces CYP3A4

P53 and PGP in drug resistance

-tumour suppressor gene

-inactivation in ~50% of cancers

-associated with drug resistance and poor prognosis

-Wt p53 represses Pgp transcription via direct DNA binding

-Wt p53 mediated downregulation of Pgp via miR-34a and LRPPRC

-mutant p53 cooperates with ETS-1 to upregulate ABCB1 expression

how does pgp mediate drug resistance

via a novel mechanism involving lysosomal sequestration

-in addition to enhanced excretion

-lysosomal accumulation of anticancer drugs as a novel mechanism

-plasma membrane containing Pgp buds inwards to form early endosomes

-as the endosome matures into a lysosome it becomes increasingly acidified

-drugs such as doxorubicin (pgp substrate) enters the cell and the lysosome and not able to reach the nucleus which is the target

-overcome with agents that increase the pH of the lysosome, due to ionisation

mechanism of drug extrusion by brain endothelial cells

via lysosomal drug trapping and disposal by neutrophils

-lysosomal sequestration in endothelial cells of the BBB

-shedding of vesicles, attached at apical side followed by phagocytosis by neutrophils

-novel mechanism of drug disposal and BBB protection

BSEP/ABCBII

-originally described as ‘sister of P-glycoprotein’

-transports bile salts so renamed Bile Salt Export Pump

-exclusively expressed in hepatocytes, mainly at canalicular membrane

-main role is to transport bile salts across canalicular membrane of hepatocyte

-patients with rare genetic disease called PFIC2 (progressive familial intrahepatic cholestasis) have inherited defect in BSEP

-could cause jaundice or an enlarged liver

-reported to transport some drugs – vinblastine

MDR3/ABCB4

-a specific translocase for phosphatidylcholine

-translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane for extraction into the lumen by bile salts

-form micelles with bile salts to protect hepatocyte biliary membrane

-genetic mutations of ABCB4 leads to 3 distinct but related hepatobiliary disease: PFIC3, gallstones and intrahepatic cholestasis of pregnancy (ICP)

-anthracyclines, vinca alkaloids, taxanes

ABCC/MRP transporters

-multidrug resistance-associated protein (MRP)

-ATP-dependent high molecular weight membrane proteins

-at least 12 different MRPs are now known

-variety of diverse functions ranging from protection from xenobiotics to channelling ions

-facilitate the extrusion of numerous glutathione, glucuronate and sulfate conjugates

-expressed in numerous tissues of the body (ubiquitous)

structure of MRP1

-has the 2 transmembrane domains and at least one additional one known as transmembrane domain 0, comprised of 5 alpha helical regions

-function may be localisation (not transport)

-additional domain makes it a larger protein

-discovered in early 90s because they were able to determine that there were lots of other cell lines or samples that were multi-drug resistant but didn’t express pgp

-encoded by ABCC1, chromosome 16

-190kDa

-main MRP member contributing to MDR

-expressed at high levels in variety of tissues including brain, testis and lung, but very low levels in liver

-preference for amphiphilic organic ions

LTC4

-leukotriene C4

-physiological high affinity substrate

-family of lipid mediators of inflammation synthesised from arachidonic acid

-LTC4 is formed by the conjugation of GSH to LTA4 through the reaction catalysed by leukotriene synthase enzyme, which is active in eosinophils, monocytes, neutrophils and macrophages

-MRP1 mediates transport of LTC4 across the plasma membrane

-KO mice have impaired inflammatory response

-formation in lung is important in asthma and allergy

what is the difference between MRP1 and other transporters

-the difference between MRP1 and other transporters is its transportation of drugs or xenobiotics that are conjugated to glutathione

-MRP1 can transport glutathione conjugates but also uses GSH as a cotransporter e.g. for vincristine

MRP-1 KO mice

-no significant difference in viability or fertility

-elevated tissue levels of glutathione (GSH) including breast, lung, heart, kidney, muscle, colon, testes, bone marrow cells, blood mononuclear leukocytes and blood erythrocytes

-unchanged tissue levels of glutathione (GSH) in organs expressing little/no MRP1 such as the liver and SI

-MRP1 is dispensable for development and growth

-however increased sensitivity to several chemotherapies

how does expression of MRP1 affect prognosis

-with a diagnosis of cancer, those that have a lower expression of MRP1 have a better prognosis than those with a higher expression

-MRP1 is a direct downstream target of MYCN in neuroblastoma

-in half of the cases of neuroblastoma there is amplification of oncogene (drives uncontrolled proliferation) – associated with poor prognosis

-MYCN1 causes upregulation of MRP1

lipid signalling and cancer

-MRP2 transports glucuronides

-high level of expression on bile canaliculus of the hepatocyte, also on apical membrane of kidney and intestine

-important contribution of elimination of drug glucuronides in bile (diclofenac, morphine, fexofenadine)

-important physiological role in elimination of bilirubin from the body

Haem metabolism and MRP2

-MRP2 transports bilirubin glucuronide from the liver to the bile

-in the rare metabolic disease Dubin-johnson syndrome, there is no active MRP2 due to mutations

-individuals with this disorder have high levels of bilirubin glucuronide in their plasma

-usually a benign condition but may see jaundice in pregnancy or with some drugs

CTFR as an ABC transporter

-CFTR is an atypical member of the ABCC family

-chloride channel which allows bidirectional diffusion of small anions

-only 2 transmembrane domains

ABCC8/SUR1

-sulphonylurea receptor important in control of blood glucose by the pancreas

-no transport role but acts as ATP-sensitive regulator of potassium channel

-sulphonylureas bind to receptor causing effect on K+(KATP) channel

-membrane potential becomes more positive opening voltage-gated Ca2+ channels, rise in intracellular calcium leads to increased insulin secretion

ABCG2/BCRP

additional contribution to drug resistance in tumours

-new resistance transporter called breast cancer-related protein (BCRP) because first identified in breast cancer cell line

-also expressed in other tissues and relevant to drug excretion

-aka MXR: mitoxantrone-resistance protein

Structure:

-single spanning transporters

-smaller that ABCB and ABCC families-only one set of membrane spanning domains (MSD)

-half-transporter

-~70kDa

-form homodimers (BCRP)(or heterodimers (ABCG5, ABCG8))

Location:

-similar location to pgp

-high levels in lactating breast

-secretion of xenobiotic into milk which has implications for breast-fed infants

-restriction on use of certain drugs by nursing mothers

inherent resistance of CML-initiating cells to imatinib

-cancer also stems from cancer stem cells

-these cells are driving the main proliferation of the tumour

-main research was done in leukaemia as they are blood cells – easier to study

-had cell surface markers of stem cells and upregulation of ABC transporters

-they were able to efflux therapy out of the cell and remain active, this cause disease relapse

-CML is a disease resulting from translocation of BCR-ABL gene; leads to an activated tyrosine kinase which can then phosphorylate various substrates and leads the signalling downstream

-imatinib was one of the first tyrosine kinases approved in cancer research

-cancer stem cells were largely resistant to imatinib treatment, mainly due to upregulation of ABCG2

ABCG2 and imatinib resistance

-its complicated

-evidence that imatinib is a substrate for ABCG2 by some studies

-CML stem cells appear to have higher levels of ABCG2 than more mature CML cells

-less sensitive to imatinib and likely to remain after mature cells eliminated

-some evidence that imatinib resistance and likely to remain after mature cells eliminated

-some evidence that imatinib resistance involves decreased levels of regulatory miRNA which results in increased ABCG2 levels in leukemic cells

-imatinib-mediated inhibition of BCR-ABL – downregulation of BCRP level post-transcriptionally via the PI3K-Akt pathway

TKIs as ABC transporter substrates/inhibitors

-RTKs are enzyme coupled receptors which mediated growth factor signalling

-TKIs targeted treatment for cancers

-targeting specific signalling pathways deregulated in cancers

-non-toxic and more specific compared with traditional cytotoxic chemotherapies but problems with acquired resistance

-important considerations especially if TKIs are used in combinations with traditional chemotherapy agents

ABCG2 and gout

-GWAS showed that a genetic polymorphism which results in an aa change in BCRP is a risk factor for gout

-accumulation of uric acid as crystals in joints occurs resulting in pain and inflammation

-further work shows that uric acid is a substrate for BCRP and unstable form of this protein results in poor ability to excrete this compound

-uric acid is generated by purine metabolism

-accumulation in the body can cause effects in joints and kidney

-can lead to symptoms like arthritis

-the mutation changes the NBD structure

-those with Asian heritage are more likely to have this snip, cells are much more resistant so have greater level of survival so can efflux the drug out

-ethnic differences should be considered during drug development phase and in clinical trial e.g. lower dose etc

strategies t overcome ABC mediated MDR

-chemical inhibitors, inhibit efflux of drugs therefore trapping a greater percentage of chemotherapeutics within the cell

-natural compounds, if they are substrates for the transporters

-targeting antibodies

-reducing expression of transporters

-agents that bypass the transporters

-novel delivery systems (nanotechnology), encapsulating drugs in nanoparticles to evade ABC on cell surface, ‘knocking down’ ABC transporters by siRNA potentially also delivered to tumour in nanoparticles

MDR1 inhibitors

-developed to overcome MDR in cancer

1. First generation

Verapamil, quinidine, amiodarone, cyclosporine A

2. second generation

valspodar, dexverapamil

3. third generation

-dofequidar, zosuquidar, tariquidar, elacridar, biricodar

progress of MDR1 inhibitors

-clinical trials have shown mixed results

-to date there is no approved inhibitor

-toxicity is still a significant issue

-MRP1 needs to transport the compounds that are conjugated to glutathione or co-transport glutathione

-presence of glutathione is very important

-could use a strategy known as collateral sensitivity, essentially where cancer cells that have developed a mechanism to become resistant to something become sensitive to something else because of this mechanism

difficulties of targeting ABC transporters

-physiological roles of ABC transporters

-ubiquitous expression – all across the body

-transporter redundancy – could inhibit one transporter but another might compensate in terms of function

-dose adjustment and monitoring when combining inhibitors with drugs with a narrow therapeutic window