Download Transporters as drug carriers and targets in the GIT

Transporters
as drug carriers and targets
in the GIT
Pascale Anderle, [email protected], IOSI
Jon Christensen, [email protected], IOSI
Overview
• Introduction
• Relevant transporters in the GIT
• Detection of new transporters using genomics
and proteomics
– Classification of transporters
– Microarray studies and transporters
• Influence of genotypes on oral drug absorption
• Transporters and cancers, especially colon
cancers
Introduction
Venter et al. 01
Important families

Peptide transporters

Nucleoside transporters

Amino acid transporters

Monosaccharide transporters

Organic cation transporters

Organic anion transporters

Monocarboxylate transporters

ABC transporters

Bile acid transporters
→ Supplementary material
Genomics and Proteomics
• Genomics
– Genomics is the study of an organism's entire genome.
• Proteomics
– Proteomics is the large-scale study of proteins,
particularly their structures and functions.
• High-throughput techniques
– For instance Microarrays, MPSS, SAGE
Introduction into Microarray Technology
Spotting:
Probes
Photolithography
Printing
Oligomers
Physical support:
Glass slide,
nylon membrane
PCR products
Sample preparation and hybridization:
cRNA or cDNA
Single-labeling or dual-labeling
Affymetrix:
Short oligo chip
Single labeling
Fluorescence or radioactivity
or
cDNA chip:
Oligos or PCR products
Dual-labeling
Classification of transporters
 Why is classification important?
 What type of classification makes sense?
 What’s typical for a transporter?
Classification of transporters
 Classification according to transport mechanism
 TC System
 Gene Ontology
 PFAM
Pumps, channels and transporters
Transporters
Secondary transporters
Membrane Transporter Proteins: Classification
Membrane Transport Proteins
Specific Carriers
Selective Channels
Primary Active Transport
ATP-powered pumps
ATPases:
P-type, F-type and ABC-type ATPases
(ABC transporters)
Primary active transport
Energy derived from hydrolysis of ATP to ADP
liberating energy from high energy phosphate
bond
Secondary Active Transport
Facilitated Diffusion
Uniporters
Glut1-5
Facilitated diffusion
Like any diffusion, transport from an area of
higher concentration to lower concentration.
Passive transport is powered by the potential
energy of a concentration gradient and does not
require the expenditure of metabolic energy
Symporters
Antiporters
Pept1
NHE
Secondary active transport
Use of energy from another source-another secondary diffusion
gradient set up across the membrane using another ion. Because this
secondary diffusion gradient initially established using an ion pump,
as in primary active transport, the energy is ultimately derived from the
same source-ATP hydrolysis.
Channels
 Transport water or specific types of ions down their concentration or electric potential
gradients
 Energetically favorable reaction
 Form protein-lined passageway across the membrane through which multiple water
molecules or ions move simultaneously at a very rapid rate—up to 108 per second
 Plasma membrane of all animal cells contains potassium-specific channel proteins
that are generally open and are critical to generating the normal, resting electric
potential across the plasma membrane
 Many other types of channel proteins are usually closed, and open only in response to
specific signals
Uniporters
 Transport is specific and saturable
 Facilitated “low resistance” diffusion:
– Down the concentration gradient
– Accelerates reaction that is already thermodynamically favored
 Reversible
 Rate much higher than passive diffusion:
- Molecule never in contact with hydrophobic core of the membrane
Uniporters: Example GLUT1
Facilitated vs. passive diffusion
Mechanism of transport
Secondary transporters
 Couple the movement of one type of ion or molecule against its concentration gradient to the
movement of a different ion or molecule down its concentration gradient
 Ability to transport two different solutes simultaneously also called co-transporters
 Mediate coupled reactions in which an energetically unfavorable reaction coupled to
energetically favorable reaction
 Catalyze “uphill” movement of certain molecules often referred to as “active transporters”, but
unlike pumps, do not hydrolyze ATP (or any other molecule) during transport
Symporters and antiporters: Examples
Pept1 and NHE
NHE
Pumps
 Use the energy of ATP hydrolysis to move ions or small molecules across a membrane against a chemical
concentration gradient or electric potential.
 Overall reaction—ATP hydrolysis and the “uphill” movement of ions or small molecules—is energetically
favorable
 P, F, and V classes transport ions only, whereas the ABC superfamily class transports small molecules as well
as ions.
Pumps: Example ABCB1
TC System: Function/Phylogenetic
 Milton Saier et al. (http://www.tcdb.org/)
 Functional/phylogenetic system
Analogous to the Enzyme Commission (EC) system for classification of enzymes, except that it
incorporates both functional and phylogenetic information. EC strictly functional.
 Designed for classification of all transmembrane transport protein found in living organisms on Earth
 Nearly 400 families
 Affiliation with a family requires rigorous statistical criteria of homology (Saier 1994). Comparison over
60 residues
TC System II: Function/Phylogenetic
1. Channels/Pores
1.A. α-Type channels
1.B. β-Barrel porins
1.C. Pore-forming toxins
(proteins and peptides
1.D. Non-ribosomally
synthesized channel
1.E. Holins
2. Electrochemical Potentialdriven Transporters
2.A. Porters
Uniporters, symporters, antiporters
2.B. Nonribosomally synthesized porters
3. Primary Active Transporters
4. Group Translocators
5. Transport Electron Carriers
8. Accessory Factors Involved in Transport
9. Incompletely Characterized Transport Systems
2.C. Ion-gradient-driven energizers
Gene Ontology Consortium
GO Output
Cellular Component
L3
L3
L4 GO:X
Molecular Function
L3
L3 GO:Y
Biological processes
L3 GO:Z
L3
L4 GO:Y
ABCB1
Two pragmatic purposes of ontology:
1. Facilitate communication between people and
organizations
2. Improve interoperability between systems
Ontologies are structured vocabularies in the form of directed
acyclic graphs (DAGs) that represent a network in which each
term may be a “child” of one or more than one ”parent”.
Human Genome Organization: HUGO
The Human Genome Organization (HUGO) Nomenclature Committee Database has as a goal to make sure that each
symbol is unique, and ensures that each gene locus is only given one approved gene symbol
In HUGO Nomenclature Committee Database:
SLC series: Currently 43 families and 298 transporter genes
Non-SLC human transport-related genes:
ATP-driven transporters
Channels
Ionotropic receptors
Aquaporins
Transporter and channel subunits
auxiliary/regulatory transport proteins
http://www.bioparadigms.org/slc/intro.htm
PFAM Database

Database of protein domain families

Contains curated multiple sequence alignments for each family, as well as profile hidden Markov
models (profile HMMs) for finding these domains in new sequences

Contains functional annotation, literature references and database links for each family

There are two multiple alignments for each Pfam family:
1. Seed alignment that contains a relatively small number of representative
members of the family
2. The full alignment that contains all members in the database that can be
detected

The profile HMM is built from the seed alignment using the HMMER package (see
http://hmmer.wustl.edu/ ), which is then used to search the pfamseq sequence database

Position specific iterative BLAST (PSI-Blast): Position specific scoring matrix (PSSM) is
constructed (automatically) from a multiple alignment of the highest scoring hits in an initial
BLAST search. The PSSM is generated by calculating position-specific scores for each position
in the alignment. Highly conserved positions receive high scores and weakly conserved positions
receive scores near zero. The profile is used to perform a second (etc.) BLAST search and the
results of each "iteration" used to refine the profile. This iterative searching strategy results in
increased sensitivity.
Transporters in the GIT
ABCB1
SLC5A1
ABCC3
SLC2A2
SLC28A1
SLC15A1
SLC2A1
SLC29A1
facilitated
Various forms of transporters and carriers working in an intestinal epithelial cell. Gluc is glucose, Nuc is nucleotide,
DP is dipeptides, ABC is ATP-binding cassette family transporter. SLC5A1 is the sodium-dependent glucose
transporter, SLC2A2 is the glucose transporter 2, SLC28A1 is the concentrative nucleotide transporter 1, SLC29A1 is
the equilibrative transporter 1 and SLC15A1 is the di/tri-peptide transporter.
Transporters as drug carriers:
Design of prodrugs
O
N
HN
H2 N
N
N
N H2
Intestinal cell
O
O
O
Valacyclovir
O
HO
N
N
H
N
N
O
O
Acyclovir
N
O
O
N
O
O
N
O
N
H
O
H
N
N
O
II. Cyclosp orin e A
Basolateral side
Apical Side
I. V al ac ic lo vi r
H
N
O
VACV
VACV
ACV
ACV
hPEPT1
P-gp
Genomics and transporters I
Profiling of the intestinal mucosa

Genes encoding proteins functioning in metabolism, transport, and cell–cell communication have the most dynamically
regulated expression profiles (Bates et al. Gastroenterolology 2002).

In contrast to Bates et al. class of transporters was not significantly regulated compared to other GO classes, however,
the subclasses carriers, and in particular its daughter classes “antiporters” and “symporters” (Anderle et al. BMC
Genomics 2005).
Profiling of in vitro systems, namely Caco-2 cells

Caco-2 cell differentiation is accompanied by coordinated down-regulation of genes involved in cell cycle progression and
DNA synthesis, which reflected the concomitant reduction in cell proliferation (Mariadason et al. Canc Res 2002).

In contrast to Mariadason et al. Fleet et al. observed in a subclone of the Caco-2 parental line, the so-called Caco-2 BBe
cells, a significant number of transporter genes being regulated upon differentiation (Fleet et al. Physiol. Genomics 2003).

Distinctive switch in gene expression patterns upon formation of cell-cell contacts. Proliferating, non-polarized Caco-2
cells more similar to human colon cancer, once differentiated more closely to normal colonocytes. Clustering identifies
“normal epithelial cluster” (genes expressed in normal coloncytes and postmitotic, polarizing Caco-2 cells) containing
SLC39A5, SLC26A2, SLC17A4 (Saaf et al. Mol Biol Cell 2007)
Genomics and transporters II
Comparison of intestinal mucosa and Caco-2 cells

Comparison of 750 genes encoding transporter and channel proteins in differentiated and undifferentiated
Caco-2 cells, human small intestine and colon indicates that Caco-2 cells consist of characteristics of
colonocytes, small intestinal enterocytes and tumor cells (Anderle et al. Pharm Res 2003).

26, 38 and 44% of 443 genes, of which 170 are transporters or channels, detectable in 4-day-old Caco-2 cells,
16-day-old Caco-2 cells and human duodenum, respectively. In vivo/in vitro drug permeability measurements
correlated well for passively absorbed drug, whereas the correlation coefficient decreased for carrier-mediated
drugs. Observed permeability of carrier-mediated drugs higher in human duodenum than in Caco-2 cells.Most
of the transporters expressed in the human duodenum also expressed in Caco-2 cells. (Sun et al. Pharm Res
2002)

In silico approach exploiting publicly available data sets (results fron 9 different labs, different platforms) in order
to assess the Caco-2 model system → Increase of robustness of resulting findings. Principal component
analysis showed that Caco-2 cells express a transport protein profile which to some extent represents the
absorptive enterocytes, but also colonocytes (Calcagno et al. Mol Pharm 2006).
Genomics and transporters III
GENOTYPE:
The genetic makeup of an individual. The fundamental constitution of an organism
in terms of its hereditary factors
PHENOTYPE:
The physical and physiological traits of an individual resulting from genotype and
environment
VARIANT:
An alteration or change in the genetic sequence.
HAPLOTYPE
A combination of alleles at multiple linked loci that are transmitted together (Greek
haploos = simple).
Genomics and transporters III
 UCSF “Pharmacogenetics of Membrane Transporters Project” (PMT,
http://pharmacogenetics.ucsf.edu/index.htm)
→ Identification of sequence variants in genes encoding selected membrane transport
proteins and the functional characterization of these variants.
 PharmGKB database (http://www.pharmgkb.org/)
→ Publicly available database that serves as a genotype-phenotype resource focused
on pharmacogenetics and pharmacogenomics.
 NCBI project (http://www.ncbi.nlm.nih.gov/SNP/)
 Relevant variants cf. Supplementary material
Transporters and diseased states of the intestine
•
Transporters as drug carriers involved in:
–
–
chemo-resistance (i.e. ability of cancer cells to become resistant to the effects of the chemotherapy drugs) and
chemo-sensitivity (i.e. susceptibility of tumor cells to the cell-killing effects of chemotherapy drugs)
•
Transporters as tumor suppressors: SLC5A8, SLC26A3, SLC1A2 (Chapman et al. , Cancer Res
2002, Li et al. PNAS 2003, Schweinfest et al. J Biol Chem 2006, Ueno et al. Tumour Biol 2004)
•
Transporters in intestinal tumor-initiating cells (stem cells, CD133+ cells): ABCG2 (O'Brien et al.
Nature 2007, Monzani et al. Eur J Cancer 2007)
•
Transporters and EMT:
–
–
•
SLC16A3 (MCT4) (Gallagher et al. Cancer Res 2007, Ho et al. 2006
Regulation of SLC15A1, CDH17 and CDX2 (Shimakura et al. , Biochem Pharmacol 2006, Nduati et al. J Biol
Chem 2007, Guo et al. Cancer Biol Ther 2004, Suh et al. Mol Cell Biol 1996, Witek et al. Clin Cancer Res 2005,
Gross et al. Oncogene 2008)
Inflammatory bowel disease: PEPT1-mediated transport of bacteria-derived n-formyl peptides such
as formyl-Met-Leu-Phe (fMLP) induces basolateral to apical neutrophil migration in a
neutrophil//Caco-2-BBE cell model (Merlin et al. J Clin Invest 1998)
Chemo-sensitivity and -resistance:
NCI Database
Scherf et al. 2000, Nature Genetics
Chemo-sensitivity and -resistance: SLCs
Sorted correlation coefficients
SLC29A1
ABCB1
Effects of expression of transporters on
dose-response curves
Role in cancer
Precarcinogen
Metabolic
activation
Carcinogen
Normal cell
Metastasis
(>1 year)
Detoxification
Secretion
Solute carriers playing a role
in EMT, invasion and
metastasis formation
ABC transporters as
efflux pumps of toxic compounds
incl. chemotherapeutics
Initiation
(1-2 days)
Invasion, EMT
(>1 year)
ABC transporters
In stem cells
Promotion
(>10 years)
Initiated cell
Progression
(>1 year)
Preneoplastic cell
Solute carriers as
tumor suppressors
Neoplastic cell
Metaplastic cell
Solute carriers as
tumor markers and as drug carriers
Summary
 What’s the relevance of transporters in oral drug absorption?
 What’s the relevance of transporters as targets in the GIT?
 What was the big break through with the development of “genomics”
technology?
 What are the limitations of “genomics” methods?
 Why is it important to know genotypes and phenotypes of variants?