Topology of ABCB1 and ABCC7 and evaluating combination therapies that treat CF Introduction
Topology of ABCB1 and ABCC7 and evaluating combination therapies that treat CF
Membrane transport, ABC transporters: general structure, subfamilies
Membrane transport is important in all living organisms.
To maintain cellular function, cells need to exchange substances with their environment, by absorbing external nutrients and secreting waste products.
Three mechanisms for membrane transport exist: passive diffusion, facilitated diffusion and active transport.
The latter two: facilitated diffusion and active transport are mediated by membrane-bound transport proteins. There are four different classes of membrane-bound transport proteins: ion channels, aquaporins, transporters and ATP-powered pumps.
ATP-binding cassette (ABC) proteins are a type of ATP-powered pump
(Vasiliou, Vasiliou and Nebert, 2008) that uses the ATP hydrolysis to translocate a range of compounds across biological membranes. The site at which ATP binds is water-soluble hence facilitating the translocation of compounds that would otherwise not pass the lipid bilayer membrane.
ABC transporters are important in a range of physiological processes and this explains why they are ubiquitously expressed. ABC transporters are present in both prokaryotes and eukaryotes. These pumps are of two types.
Influx transporters move substrates in the cell and efflux transporters move substances outside the cell. In bacteria, both influx and efflux transporters are found but most eukaryotic ABC transporter are efflux. In mammals like us, ABC transporters are mostly expressed in the intestine, kidney, liver, blood-brain barrier and placenta (Vasiliou, Vasiliou and Nebert, 2008).
General structure of ABC transporters
ABC transporter proteins are structurally complex and span the plasma membrane multiple times. They are made up of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD).
The highly conserved primary structure of the ATP-binding domains includes the presence of a phosphate-binding loop (P-loop or Walker A motif) and a short consensus sequence “LSGGQ” that is involved in nucleotide binding.
These regions can be found on the same protein or on two different ones.
Most ABC transporters function as a dimer and therefore are constituted of four functional domains: two nucleotide-binding domains (NBDs; NBD1 and NBD2) and two transmembrane domains (TMDs; TMD1 and TMD2) that are interconnected by intracellular (ICLs) and extracellular loops (ECLs). Each TMD contains six transmembrane segments. In eukaryotes, many ABC transporters, all four of these functional units exist on a single polypeptide” (Wilkens, 2015).
Based on structural similarities, ABC transporters can be categorized into subfamilies. In humans for example, there are 48 ABC transporters divided in eight subfamilies (Wilkens, 2015).
Many of these human ABC transporters have been associated with disease states including adrenoleukodystrophy, cancer and cystic fibrosis (Wilkens, 2015).
ABCC7, for example, is a cystic fibrosis transmembrane regulator (CFTR) protein
so if this protein becomes dysfunctional, it could lead to cystic fibrosis.
In this essay, the topology of CFTR will be compared with another ABC transporter, ABCB1, known for its role in multi-drug resistance.
Structure of ABCB1
ABCB1 is a multi-drug transporter P-glycoprotein (P-gp) and will be referred to as P-gp in this essay. This efflux pump is composed of “170-kDa transmembrane protein that is N-glycosylated at the first extracellular loop” (Hamidovic, Hahn and Kolesar, 2009). ABCB1 has two hydrophobic transmembrane domains that dimerise to form a pore. The pore plays important role in translocating substrates across the membrane and contains highly conserved residues that can recognize a range of different substrates. The extracellular-facing portion of pore is lined with hydrophobic amino acids whereas cytosolic-facing half of pore contains polar-charged residues (Hodges et al., 2011). Substrate binding happens on the cytosolic facing half of pore.
The protein is highly flexible and conformationally changes shape to bind and export substrates (Hodges et al., 2011).
Drug binding of P-gp
Recent experiments suggest that the drug binding sites of P-gp reside in a
“funnel shaped binding pocket” (Sauna and Ambudkar, 2007) where multiple helices from both TMDs form overlapping drug-binding sites.
For ABC transporters, whether importers or exporters, transport substrate needs to interact “with residues of the transmembrane ?-helices that line the transmembrane pore” (Wilkens, 2015). However, ABCB1 is different. Recent experiments have identified multiple helices from both TMDs forming several overlapping drug-binding sites. Consequently, the drug-binding pocket of P-gp was characterised as being ‘polyspecific’ towards its transport substrates (Wilkens, 2015).
This explains why ABCB1 can confer ‘multi-drug’ resistance.
P-gp ATP-binding and mechanism of efflux
P-gp has two independent yet coupled functions: substrate transport and ATP hydrolysis. Conformational changes at the NBDs cause conformational changes in the drug-binding site. The crucial feature of the nucleotide-binding pocket is that
“the ATP is sandwiched between the Walker A, Walker B, Q-loop, and H-loop of one NBD and the D-loop and signature sequence of the apposing NBD, hence the term “ATP sandwich” (SHEPPARD and WELSH, 1999). Figure 1 shows a schematic.
The transport event is described below.
The binding of drug and ATP initiates ABCB1 transport pathway.
Since this protein is an exporter, the drug binds to protein at cytosolic-facing portion of pore with high affinity. Upon ATP binding, the protein undergoes a conformational change at the NBDs. This conformational change is transmitted from the NBDs to the TMDs causing the protein to transform itself into a low-affinity extracellular-facing site, thereby exporting the drug (Sauna and Ambudkar, 2007).
The pump then resets (SHEPPARD and WELSH, 1999).
P-gp recognizes and exports a large variety of both hydrophobic cytotoxic and
non-cytotoxic drugs (Hamidovic, Hahn and Kolesar, 2009).
These include endogenous compounds, xenobiotics and compounds that modulate P-gp activity (Hodges et al., 2011). Compounds that bind P-gp can behave as both substrates and inhibitors.
Cellular and tissue distribution of ABCB1
The cells in which P-glycoprotein is expressed at the membrane include the elimination and barrier organs, “where it has protective and excretory functions” (Hodges et al., 2011).
P-gp plays a role in greatly reducing the concentration of orally administered drugs before they reach the systemic circulation by effluxing drugs from the “lumen-facing epithelia of the small intestine and colon, and from the bile-facing canaliculi of the liver” (Hodges et al., 2011) allowing the drug to be removed by biliary excretion.
P-gp is also expressed at the “urine-facing side of the brush border membrane of proximal tubules in the kidney” (Hodges et al., 2011).
This means that even if drug has reached the systemic circulation already,
P-gp can eliminate it by exporting the drug from the proximal tubules into the urine.
P-gp is also expressed in lymphocytes and other immune and blood components where it is involved in trafficking cytokines and viral resistance (Hodges et al., 2011).
P-gp is also expressed in adrenal cortex suggesting that it could contribute to glucocorticoid resistance, hormone transport and homeostasis (Hodges et al., 2011).
ABCC7 structure – comparing with P-gp (ABCB1)
ABCC7 is the cystic fibrosis transmembrane regulator (CFTR) and is a chloride channel. Whilst CFTR and P-gp are both comprised of two membrane-spanning domains (MSDs) and two NBDs, CFTR has also has a fifth domain called the regulatory (R) domain. The MSDs form the Cl- selective channel pore.
The R domain contains multiple phosphorylation sites. R domain phosphorylation determines CFTR channel activity (SHEPPARD and WELSH, 1999).
NBDs hydrolyse ATP to control channel gating. Figure 2 shows a model of ABCC7.
CFTR drug binding
Through cry-electron microscopy techniques, the drug binding pocket of CFTR was deduced to be at the C-terminal region of MSDs and comprised of residues from different domains within the interface between NBD1 and intracellular loop 4 (IL4) (Molinski et al., 2018).
There are more than 270 CFTR mutations. The Phe508 deletion mutation is the most common and results in cellular degradation of the protein.
When two copies of the mutation are inherited, it results in severe dysfunction of the protein (Rowe et al., 2017). CFTR protein is a tunnel shaped and has a gate that opens when chloride needs to get through. This gate is locked in the closed position when you have a gating mutation and chloride cannot flow through.
Residual function mutations account for about 5% of all patients with cystic fibrosis These mutations retain partial expression of normal CFTR so do not result in complete loss of transport ability but ‘residual’ CFTR transport as well as variably preserved channel gating and function (Rowe et al., 2017).
For different mutations, different drugs have been developed.
CFTR potentiators such as ivacaftor, treat gating mutations and can improve ion transport by increasing the probability of CFTR channel opening at the cell surface. CFTR correctors “increase the amount of functional CFTR at the cell surface” (Rowe et al., 2017) by improving the “cellular processing and trafficking of normal and mutated CFTR protein” (Rowe et al., 2017).
Cellular and tissue distribution of CFTR compared with P-gp
In humans, CFTR protein is expressed in epithelial cells of the lungs, sweat glands, gut, pancreas, in neurons of the human brain and other tissues (Guo et al., 2009).
Unlike P-gp it is not expressed in liver.
It is expressed in ciliated cells of lower airway epithelia and in non-ciliated cells of the tracheal epithelium (Hahn et al., 2017).
CFTR – ATP binding and mechanism of gating
For ATP to be biologically active, it needs to bind to a magnesium ion.
Thus, what is described as ATP is, magnesium-ATP (Mg-ATP).
There are highly conserved sequences at the NBDs of CFTR that are thought to bind and hydrolyse Mg-ATP. The sequences include Walker A, Walker B and LSGGQ motifs.
According to structural and functional studies of ATPases, the lysine residue of Walker A interacts with the phosphate of ATP and is required for ATP hydrolysis (SHEPPARD and WELSH, 1999). Walker B aspartate however, is required for ATP binding and coordinates the magnesium ion in Mg-ATP. Many mutations associated with cystic fibrosis are in these domains hence these domains are important (SHEPPARD and WELSH, 1999).
The gating behaviour of CFTR according to kinetic studies involves one open and two closed states. Data from the function studies pose that ATP controls channel gating via an interaction that increases the rate at which the CFTR transitions “from a long-lived closed state (C1) to a bursting state” (SHEPPARD and WELSH, 1999) in which the channel switches and flutters back and forth “between an open state and short-lived closed state” (SHEPPARD and WELSH, 1999).
CFTR potentiators like ivacaftor treat gating and residual function mutations and enhance opening of channel. Pharmacokinetic and pharmacodynamic studies reveal ivacaftor is an efficacious CFTR drug. The data is presented below.
Pharmacokinetics of CFTR potentiator ivacaftor: ADME
When ivacaftor is orally administered, it is readily absorbed from the gut but is not very soluble in water, with less than 0.05 ?g/mL (Committee for Medicinal Products for Human Use (CHMP), 2014).
Up to 250mg, does/time pharmacokinetics conforms to a linear profile but when the dose reaches 375mg or higher, Cmax plateaus (Committee for Medicinal Products for Human Use (CHMP), 2014)
“Ivacaftor is transported in the plasma highly bound (99%), preferentially to alpha-1-acid glycoprotein” (Fohner et al., 2017). It is also bound to its site of action located at the apical membrane of epithelial cells.
Ivacaftor is metabolized in liver by cytochrome P450 into a metabolite called hydroxymethyl-ivacaftor (M1) and an inactive metabolite ivacaftor-carboxylate (M6) (Fohner et al., 2017).
The bile is where most of the parent ivacaftor drug and its metabolites is eliminated (87%), “with 22% being the M! metabolite and 43% being the M6 metabolite”
(Fohner et al., 2017).
Whilst the M6 metabolite is eliminated via biliary excretion via the “solute carrier organic anion transporter 1B1 (SLCO1B1)” (Committee for Medicinal Products for Human Use (CHMP), 2014), it is not known how M1 is eliminated.
Ivacaftor has a half-life of 12-14 hours (Committee for Medicinal Products for Human Use (CHMP), 2014).
Pharmacodynamics of ivacaftor
Ivacaftor is used in CF patients who carry at least one of 11 so-called ‘gating’ genetic variants. These variants prevent cAMP-mediated activation of the CFTR chloride channel at the apical cell membrane and thereby also impedes normal movement of chloride (Kulczycki, Kostuch and Bellanti, 2002).
Ivacaftor is thought to stabilize the open state of CFTR channel, permitting chloride transport, but the mechanism by which it does this is not known.
Proposed mechanisms include “increasing the ATP-dependent opening rate and slowing the closing rate” (Kopeikin et al., 2014) and “decoupling the gating cycle and ATP hydrolysis cycle” (Jih and Hwang, 2013).
In both CFTR with gating mutations and CFTR with normal function, ivacaftor improves function upon binding to the protein in the epithelial cell membrane.
In CF patients with at least one CFTR gating variant, treatment improved lung function by more than 10% and an improvement in CF symptoms was observed.
In clinical trials with 6-11 year olds, ivacaftor improved weight gain and decreased chloride concentration compared to baseline.
The need for combination therapy
However, when ivacaftor was used in patients homozygous for F508del variant in clinical trials, no improvement was observed. F508 del variant is not a gating a variant but a deletion variant where a single phenylalanine residue is deleted from the amino acid sequence making up CFTR. Without this residue, CFTR cannot stay in the correct 3D shape. The cell recognizes the CFTR is in incorrect shape and destroys it. But in 2015, a combination drug of lumacaftor and ivacaftor was approved by the European Medicines Agency (EMA) and US Food and Drug Administration (FDA) for treatment in patients homozygous for the F508del allele.
Lumacaftor-ivacaftor combination therapy in patients homozygous for F508del
In lower airway epithelial cells derived from patients homozygous for the F508del allele, lumacaftor is found to restore CFTR function to about 15% of normal (Fohner et al., 2017).The F508del allele causes CFTR misfolding. The mechanism by which lumacaftor restores function is by improving the folding of CFTR and helping it localise to the cell membrane (Brewington, McPhail and Clancy, 2015).
The F508del CFTR folding improvement from lumacaftor-ivacaftor combination therapy results in a “30% improvement of CFTR function in lower airway epithelial cells derived from individuals homozygous for the F508del allele” (Brewington, McPhail and Clancy, 2015).
Lumacaftor-ivacaftor combination therapy in homozygous and heterozygous for F508del
It was proposed that improving CFTR function to clinically meaningful levels, required a combination of lumacaftor to correct misfolding and traffic CFTR to membrane and ivacaftor to enhance opening of channels at the membrane.
Consequently, lumacaftor moved to clinical trials in patients that were homozygous and heterozygous for the Phe508del mutation, so that its safety and efficacy alone and in combination with Ivacaftor could be evaluated.
In a phase 2 trial, lumacaftor alone did not provide any clinically significant benefit “as predicted FEVM1 % was similar between the studied groups” (Margarida Matos and Matos, 2018).
Following this, Lumacaftor and ivacaftor combination therapy was investigated in a series of clinical trials. Data from a phase 2 trial showed combination of Lumacaftor and Ivacaftor at higher doses improved FEV1 significantly by a mean of 6% for patients homozygous for Phe508del. It decreased pulmonary exacerbations and reduced sweat chloride concentration by a mean of 8.9 to 10.3 mmol/L in the treatment groups (Margarida Matos and Matos, 2018).
However, in Phe508del CFTR heterozygous patients there was no significant improvement in FEV1 or any other parameters.
In two phase 3 trails, patients homozygous for Phe508del showed that experience clinical meaningful improvements in FEV1, ranging from a mean of 2.6 to 4.0%.
Moreover, “the rate of pulmonary exacerbations was 30 to 39% lower” (Margarida Matos and Matos, 2018).
Why this combination therapy fell short
Even though these results were significant, they did not meet initial expectations of therapy. Experimental evidence explained that the reason these results fell below expectations was because chronic administration of Ivacaftor and other CFTR potentiators “resulted in a dose-dependent reversal of Lumacaftor- and Tezacaftor-mediated CFTR correction in Phe508del homozygous primary airway cell cultures” (Margarida Matos and Matos, 2018).
This dose-dependent reversal of CFTR correction occurred because of protein destabilization and increased turnover rate (Margarida Matos and Matos, 2018).
This led to less CFTR protein being functionally expressed at cell surface.
Another study showed that when ivacaftor was administered at high concentrations, (>1 ?M), ivacaftor suppressed lumacaftor’s ability to correct Phe508del-CFTR. However, when ivacaftor was administered at “low clinically relevant concentrations” (Margarida Matos and Matos, 2018) ivacaftor did not inhibit lumacaftor.
Having said that, combination therapy proved to be more efficacious than either ivacaftor and lumacaftor alone therefore the inhibitory effect of ivacaftor on lumacaftor requires further investigation.
Despite the combination therapy being modestly efficacious, the trial data showed therapeutic benefit for patients so was approved for clinical treatment of CF in patients homozygous for Phe508del mutation by the FDA and the EMA
(Margarida Matos and Matos, 2018).