o&e2_membrane protein-mediated cytotoxicity
TRANSCRIPT
#An Original & Easy Approach to…
This is a series of ideas with basis intended
to provide simple solutions to assess an hipothesys.
Some may result false, or but if at least one works… So, if they are interesting to you, great! Should you have any comment, just contact me or post it Should you want anything else, hire me
It’s about
#An Original & Easy Approach to…
Studying
Membrane protein-mediated cytotoxicity
Are the tools/assays we are using to filter out potential cytotoxic hits appropriate? Could we easily explode cell transportome specificity for
chemosensitivity?
The cell membrane has large content of proteins, typically around 50% of membrane volume. These proteins are important
for the cell as responsible of several biological activities including active transport: The cell membrane is selectively
permeable and able to regulate what enters and exits the cell, thus facilitating the transport of materials.
Systematic assessments of transport-cargo relationships in yeast have
uncovered a pronounced dependency on active transport mechanisms of
most tested small-molecule agents
(Winter et al, Nature Chemical Biology 10,768–773(2014))
Hypothesis:
by ‘changing’/interfering with the proteins expressed in the surface of the cell it is possible to
modify its sensitivity to a small molecule (that acts inside the cell, not at membrane level)
E.g.: inhibiting a membrane protein (channel, transporter, etc) you may change the sensitivity of a cell to a toxic cpd.
Approach: using a single cell line, try combos of a toxic cpd and inhibitors specific against
membrane proteins
Conceptually similar to programs focussing on efflux pump inhibitors, but here doing on transporters that actively bring
molecules in the cell.
Membrane protein-mediated cytotoxicity
Background and hypothesis
Objectives:
Increase knowledge of main active transport mechanisms and proteins relevant for the
internalization of small molecules. Ideally build SAR on molecule type vs potential transport
protein.
Assess the relevance of cellular assays to filter out cpds because of their toxicity against
‘standard’ cell lines (i.e. HepG2, HEK293) that may not be very representative for a particular
target
Should Probe Of Concept be met, evaluate ‘rescue’ of failed molecule (hit/lead not progressed
because of undesired toxicity)
Appraise the validity of a synergistic approach to modulate toxicity
Additional reading:
Birsoy, K. et al. MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors. Nat. Genet. 45,
104–108 (2013).
Reiling, J.H. et al. A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key
mediator in the response to tunicamycin. Proc. Natl. Acad. Sci. USA 108, 11756–11765 (2011).
Lanthaler, K. et al. Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast. BMC
Biol. 9, 70 (2011). CAS
Dobson, P.D. & Kell, D.B. Carrier-mediated cellular uptake of pharmaceutical drugs: an exception or the rule? Nat. Rev. Drug Discov. 7,
205–220 (2008).
Membrane protein-mediated cytotoxicity
Additional reading and Extensions
Membrane protein-mediated cytotoxicity
– Use combinations of ‘cidal’ molecule + set of compounds specifically inhibiting membrane
proteins (channels, carriers, etc) to be able to match loss of the cidal effect with
inhibition of a particular membrane protein.
Define cell line: e.g. HepG2
Define cidal compound(s)
Cidal will act at intracellular level [DNA, ribosome, etc])
Define inhibitors set
Set up viability assay
Analyze output
Restrictions: To reduce complexity, constrains are defined
Iterative process from low to highly specific inhibitors.
Prioritize Secondary active transport (e.g. SLC family)
Methodology (a)
Membrane protein-mediated cytotoxicity
– Opposingly to a), use set of compounds shown to be cidal for a cell type + specific inhibitor
of a carrier protein, then establish relationship between loss of the cidal effect &
inhibition of a particular membrane protein.
Define cell line: e.g. HepG2
Define cidal compounds:
Molecules annotated as NFI for their cytotoxic effect
Define ‘carrier’ inhibitor molecule(s)
Set up viability assay
Analyze output
Restrictions: To reduce complexity, constrains are defined
Iterative process from low to highly specific inhibitors.
Prioritize Secondary active transport (e.g. SLC family)
Methodology (b)
Membrane protein-mediated cytotoxicity
Additional info
Sources:
– The Handbook of Receptor Classification and Signal
Transduction
– TransportDB
Membrane protein-mediated cytotoxicity
Additional info
