P-glycoprotein pamphlet: iteration 4 of 4 final

P-glycoprotein Inhibition: A Novel
Treatment for Glioblastoma
c
What will you gain from this pamphlet?
• What p-glycoprotein is and what it looks like
• What glioblastoma is and why novel treatment methods
are important
• How understanding protein structure can benefit research
for therapeutic treatments for glioblastoma
• How to make meaningful use of research papers to justify
research
Contents
How to use guide…………………………………………...2-3
1. Introduction……………………………………………….4-6
1.1 Introduction to glioblastoma.……………………4-5
1.2 How is P-glycoprotein implicated in
glioblastoma?............................................................6
2. What is P-glycoprotein?............................................7-10
2.1 What does P-glycoprotein look like?.................7-8
2.2 How does P-gp bind substrates.......................9-10
3. Inhibiting P-glycoprotein……………………………..11-17
3.1 Models of inhibition…………………………….11-12
3.2 Examples of P-gp inhibition…………………...13-17
(3.2.1 Nb592…………………………………13-14)
(3.2.2 Tariquidar……………………………..15-16)
(3.2.3 Literature Search 1…………………..16-17)
4. Application to glioblastoma treatment…………….18-20
(4.1 Ko143…...............................................................18)
(4.2 Literature Search 2…………………………….19-20)
5. Conclusions and key take aways………………………21
Reference List…………………………………………….22-23
1

How to Use Guide
2 3
This pamphlet has been designed as an independent learning tool,
that explains why understanding what proteins look like is important
to scientific development.
There are 4 main sections to the pamphlet which contain relevant
papers with associated questions about the topic. Your task is to
work through this pamphlet – at your own pace – and answer the
questions in each chapter.
Each page has one or more scientific papers from which you can
extract the answers to questions. Use the referenced bibliography
entry to search for the relevant paper; to access full papers, you
may need to authenticate via OpenAthens. If you need
guidance on how to do this, you can find directions here:
https://library.leeds.ac.uk/downloads/download/198/open-athens-
bookmarklet-quick-start-guide
At the end of each chapter, there is an associated podcast
recording which can be accessed via OneDrive. The recordings will
provide answers for the questions in the chapter, as well as giving a
fuller explanation of the topic.
To make the most out of this resource:
1. Read the papers provided, focussing on the sections
highlighted
2. Try your best to answer all the questions before listening to the
recordings
3. When literature searching in chapters 4 and 5, use the
University of Leeds websites (linked on page 17) to help you
The application of skills will develop as you progress through the
pamphlet. Completing the tasks in order will be the most beneficial
for your learning.
There is a OneDrive file named ‘P-glycoprotein Inhibition: A
Novel Treatment for glioblastoma AUDIO FILES’ which contains
4 subfiles. They should appear as shown below.
In each folder, there is an audio file that is named after the page in
the chapter it explains. For example, the files in chapter 1 are
shown below, and align directly with the name on the associated
page.
Although there is no time frame in which this pamphlet
must be completed, a recommended time to spend on
tasks is located next to this icon on each page.
Remember this is a guide, not a limit!

4
(1.1) Introduction to Glioblastoma
Glioblastoma (GBM) is an aggressive form of a malignant, solid brain
tumour. P-glycoprotein (P-gp) is a transmembrane protein relevant to the
progression of this cancer. There are many types of cancer that require
improvement in treatment, so why focus on glioblastoma?
Use the papers presented below to provide yourself with an introduction to
what glioblastoma is and why it should be studied. Paper 1 describes
what glioblastoma is, details surrounding its diagnosis and
prognosis and current treatment methods.
References
(Paper 1) Rajaratnam, V., Islam, M.M., Yang, M., Slaby, R., Ramirez, H.M. and Mirza,
S.P. 2020. Glioblastoma: Pathogenesis and Current Status of Chemotherapy and Other
Novel Treatments. Cancers. 12(4).
(Paper 2) Chaichana, K.L., Zadnik, P., Weingart, J.D., Olivi, A., Gallia, G.L., Blakeley, J.,
Lim, M., Brem, H. and Quiñones-Hinojosa, A. 2013. Multiple resections for patients with
glioblastoma: prolonging survival. Journal of Neurosurgery. 118(4), pp.812–820.
10-15 mins
Paper 2 highlights a key method of glioblastoma treatment and
evaluates its effectiveness.
Using the results and discussion from paper 2:
5. Determine how effective current treatments for GBM are. Use
quantitative data to support your claim.
Use the abstract and introduction in this paper to answer:
1. How common is glioblastoma in relation to other brain cancers?
2. What is the average prognosis for glioblastoma after diagnosis?
3. How do your answers from questions 1 and 2 support the idea that
glioblastoma research should be prioritised?
4. What is the primary treatment method for GBM – if chemotherapy is
used, what type is usually administered?
5

7
6
(1.2) How is P-gp Implicated in Glioblastoma?
Why is P-gp relevant to the treatment of glioblastoma? A common issue
associated with cancer recurrence is multidrug resistance (MDR) – P-gp
is directly implicated in this process.
Paper 3 describes the action of P-gp in cells and how its expression
is altered in cancers.
Use the discussion section (particularly in section 5) to determine:
6. What P-gp is and how its expression is altered in glioblastoma.
7. i. How its expression affects the delivery of chemotherapeutic drugs
to a tumour.
ii. How the delivery of chemotherapeutic drugs might be altered
if P-gp is removed (knocked down).
References
(Paper 3) Heming, C.P., de Souza Barbosa , I., Miranda, R.L., Ugarte, O.N., de São
José, V.S., Moura-Neto, V. and Aran, V. 2025. P-Glycoprotein Drives Glioblastoma
Survival and Chemotherapy Resistance. American Journal Of Pathology.
10 mins (2.1) What Does P-gp Look Like?
As shown in figure 1, proteins can have a range of complex structures
which will often indicate their function. Below there are crystal structures of
3 different proteins; despite being comprised of the same components (α
helices and β pleated sheets) their quaternary structures look strikingly
different.
Paper 4 clarifies what is known about the 3D structure of P-gp using
cryo-electro microscopy.
A
B
C
Figure 1. A comparison of the crystal structures of 3 different proteins. Panel A depicts
ToMV-Hel–Tm-1(431) complex (tobacco mosaic virus inhibitor) (Ishibashi et al., 2014);
panel B illustrates P-gp’s crystal structure (Mora Lagares et al., 2022); panel C shows the
structure of the vascular endothelial growth factor receptor (Muller et al., 1997).
15 mins

Using the introduction and figure 1 from paper 4 highlight:
7. What 2 key types of domain are present in P-gp.
8. Where each domain is located relative to the cell membrane.
9. What features characterise the different domain types.
10. How the domains are connected.
References
(Paper 4) Mora Lagares, L., Pérez-Castillo, Y., Minovski, N. and Novič, M. 2022. Structure–
Function Relationships in the Human P-Glycoprotein (ABCB1): Insights from Molecular
Dynamics Simulations. International Journal of Molecular Sciences. 23(1), p.362.
(2.2) How Does P-gp Bind Substrates?
As discussed in paper 4, P-gp is polyspecific (can bind a range of
different substrates). Many proteins bind via the ‘lock and key’ hypothesis
as shown in figure 2, (A and VA, 2018) but this is not the case for P-gp.
9
Paper 5 explores the presence of drug binding sites on P-gp and
their effect on one another. Paper 6 builds on this further,
identifying the effects of certain substrates on P-gp’s function.
Using the abstract and results sections of (focus on pages 131-133)
paper 5, describe:
11. The main conclusions drawn from the paper regarding the binding
sites of P-gp.
Figure 3. An illustration of the ‘lock and key’ model of protein activation. When the substrate
(navy square) binds to the protein (blue ¾ circle), this initiates a conformational change. Figure
created using PowerPoint.
A
B
C
8
20 mins
Figure 2. A figure adapted from Condic-Jurkic et al. (2018) highlighting the simple cycle of P-
gp from an open to a closed conformation.

10
Using the abstract and results (focusing on figures 2 and 3) sections of
paper 6, determine:
12. What XR9576 is and how it affects the function of P-gp.
13. What the different classes of binding sites are (based on the action of
XR9576).
Using what you have learnt so far, consider the similarities and
differences between the substrate binding process shown in figure 2 and
P-gp.
References
(Paper 5) Shapiro, A.B. and Ling, V. 1997. Positively Cooperative Sites for Drug Transport by
P‐Glycoprotein with Distinct Drug Specificities. European journal of biochemistry. 250(1),
pp.130–137.
(Paper 6) Martin, C.A., Berridge, G., Mistry, P., Higgins, C.F., Charlton, P. and Callaghan, R.
1999. The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein.
British Journal of Pharmacology. 128(2), pp.403–411.
(3.1) Models of P-gp Inhibition
Based on the structure of P-gp, there are two basic theoretical mechanisms
by which it could be inhibited. Using the knowledge you have gained so far,
try filling in the diagram below that illustrates these two processes of
inhibition.
Paper 7 outlines the transport cycle of P-gp.
Using the introduction and figure 1a from paper 7, as well as your answers
to questions 7-12, fill in the diagram below.
12-15 mins
11

14. Describe and explain the process of inhibition you can see in:
i. Cycle A
ii. Cycle B
iii. Why is it integral that the structure of P-gp is understood to
suggest the methods of inhibition shown?
12
(3.2.1) P-gp Inhibition: Nb592
13
Based on the models shown on page 11, paper 8 defines one
example of P-gp inhibition using nanobody 592 (Nb592). This is
illustrated by development of 3D crystal structures of P-gp in the
inward facing conformation.
Using the introduction and results sections (use figure 2 to help
you) of paper 8:
15. Define what a nanobody is. Why might nanobodies make good
drugs?
(If you are interested in nanobodies or would like a more detailed
explanation of what they are, refer to Bao et al. (2021), which will be
indicated in the reference list).
References
(Paper 7) Condic-Jurkic, K., Subramanian, N., Mark, A.E. and O’Mara, M.L. 2018. The
reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a
membrane environment. PLOS One. 13(1), p.e0191882.
Figure 4. Illustration of a classical antibody (B) in comparison to a heavy chain antibody
(C). The green section on in panel C illustrates the nanobody, a structure relevant to
potential P-gp inhibition. Figure taken from Bao et al. (2021)
A B
15 mins

16. Explain where Nb592 binds to P-gp.
17. Which path of inhibition does Nb592 binding initiate? Refer to the
diagram on page 11.
18. Explain how Nb592 causes the selected inhibition pathway.
References
(Paper 8) Ward, A.B., Szewczyk, P., Grimard, V., Lee, C.-W. ., Martinez, L., Doshi, R.,
Caya, A., Villaluz, M., Pardon, E., Cregger, C., Swartz, D.J., Falson, P.G., Urbatsch, I.L.,
Govaerts, C., Steyaert, J. and Chang, G. 2013. Structures of P-glycoprotein reveal its
conformational flexibility and an epitope on the nucleotide-binding domain. Proceedings of
the National Academy of Sciences. 110(33), pp.13386–13391.
15
(3.2.2) P-gp Inhibition: Tariquidar
One of the more promising inhibitors of P-gp that has been studied in
vitro is tariquidar. The effect of this drug on P-gp was explored in
paper 9, which describes how it holds P-gp in a closed
conformation.
Using the written and graphical abstracts as well as the discussion
in paper 9, determine:
19. Which inhibition cycle does tariquidar represent (in reference to the
diagram on page 11)?
20. How is P-gp held in a closed conformation? (Consider where drug
binding occurs).
21. How does holding P-gp closed stop its transport cycle?
20 mins
Despite tariquidar showing promising inhibition of P-gp in vitro, in vivo
studies did not have the same success. Paper 10 explores why
tariquidar does not make a good drug, and how researchers have
refined its structure to make it more ‘drug like’.
14

17
To help you with this task, look at how the use of specific papers have
been justified throughout this pamphlet.
Literature Search Help
• University of Leeds 2024.
Literature searching explained.
Leeds.ac.uk. [Online]. Available
from:
https://library.leeds.ac.uk/info/1
404/literature-
searching/14/literature-
searching-explained.
• University of Leeds 2025.
Leeds Harvard referencing
examples | Study and research
support | Library | University of
Leeds. library.leeds.ac.uk.
[Online]. Available from:
https://library.leeds.ac.uk/refere
ncing-examples/9/leeds-
harvard.
30 mins
Using the introduction and conclusion sections of paper 10, highlight:
22. Why tariquidar does not act in a ‘drug like’ manner.
23. How the researchers altered tariquidar and what effect this had.
References
(Paper 9) Loo, T.W. and Clarke, D.M. 2014. Tariquidar inhibits P-glycoprotein drug efflux but
activates ATPase activity by blocking transition to an open conformation. Biochemical
Pharmacology. 92(4), pp.558–566.
(Paper 10) Teodori, E., Dei, S., Bartolucci, G., Perrone, M.G., Manetti, D., Romanelli, M.N.,
Contino, M. and Colabufo, N.A. 2017. Structure-Activity Relationship Studies on 6,7-
Dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline Derivatives as Multidrug Resistance
Reversers. ChemMedChem. 12(16), pp.1369–1379.
The development and modification of tariquidar into more ‘drug like’
compounds relies on understanding what P-gp looks like. Using what you
have learned from papers 9 and 10, complete a literature search (3.2.3)
for a paper that builds on their conclusions.
Consider how minor alterations to the original compound led to an
improved outcome – think about using key works in your search such as
‘analogue’ and ‘bioisosteres’.
Using your chosen paper:
24. Explain what it covers and justify its relevance to the task in 1-2
sentences.
25. Identify which areas of the paper were most useful for retrieving the
information you wanted.
26. Create a bibliography entry for your paper using the Leeds Harvard
system.
16

18 19
(4.1) Application to Glioblastoma Treatment:
Ko143
We have now explored how P-gp can be inhibited, but how is this being
applied to treating glioblastoma?
Paper 11 illustrates application of a P-gp inhibitor in the context of
glioblastoma regarding improved TMZ delivery.
Use paper 11 to determine:
27. What is the ‘starting drug’ this paper is based on? Why was this drug
chosen to be developed?
28. How effective was the tested drug? Can you find a figure in the paper
to support your claim?
References
(Paper 11) Lustig , S.D., Kodali , S.K., Longo, S.L., Kundu, S. and Viapiano, M.S. 2022.
Ko143 Reverses MDR in Glioblastoma via Deactivating P-Glycoprotein, Sensitizing a
Resistant Phenotype to TMZ Treatment. Anticancer Research. 42(2), pp.723–730.
(4.2) Application to Glioblastoma Treatment:
Other Avenues?
There are few P-gp inhibitors that have been developed and are applied
to glioblastoma treatment, primarily due to pharmacokinetic and dynamic
issues.
29. Find a piece of research (4.2) that explores another avenue related
to P-gp and multidrug resistance. (Consider how altering the
expression of P-gp could affect TMZ delivery.)
30. Why is an understanding of what P-gp looks like still relevant to this
research?
You have not been given specific areas of
the paper to focus on when answering these
questions. Have a think about which
sections of the papers have been most
referred so far in this pamphlet to help you.
Protein engineering
Expression
Post-translational
modification
Inhibition
The protein structure shown above was made using BioRender’s Protein
Data Bank (PDB) Builder – create your own images accessing BioRender
here: https://www.biorender.com/
For a more detailed look, use the PBD ID of proteins to view structures in
the Protein Data Bank itself where you can explore different modelling
types, protein densities and conformations: https://www.rcsb.org/
20 mins 30 mins

20 21
Congratulations! You have made it to the end of this pamphlet.
Hopefully, you feel that this was a useful learning tool, and you are
more informed about why understanding protein structure is so vital
to biomedical research.
To summarise, from this resource you should now have a greater
understanding of:
• What glioblastoma is and why developing treatments for GBM
is important
• How P-gp is involved in multidrug resistance and how this can
impact the treatment of GBM
• Why understanding what P-gp looks like is vital to
developing treatments based around this protein
• What current treatments have been developed and how a
structural understanding of P-gp helped in their development
• How to use scientific papers in a streamline and
straightforward manner
• How to search for scientific papers in a streamline and
straightforward manner
Introduction to the Protein Data Bank
These images of P-gp are shown in
the Protein Data Bank using ID
3G60, illustrating P-gp’s polyspecific
binding ability. There are a number
of different conformations you can
view P-gp in, using different ID
codes. These can be found with a
quick internet search!
You can specify how you want the protein and/or
bound ligands to be presented (ball and stick,
surface display) using the panel shown on the
left. You can also highlight and remove areas,
drawing attention to specific structures, bonds
and complexes.
For further information about the PBD and its
influence on scientific research, access the
PBD 101 page here: https://pdb101.rcsb.org/
Conclusions and Key Take-aways

22 23
A, T. and VA, B. 2018. Molecular Docking: From Lock and Key to Combination
Lock. Journal of Molecular Medicine and Clinical Applications. 2(1).
Bao, G., Tang, M., Zhao, J. and Zhu, X. 2021. Nanobody: a promising toolkit for
molecular imaging and disease therapy. EJNMMI Research. 11(1).
Chaichana, K.L., Zadnik, P., Weingart, J.D., Olivi, A., Gallia, G.L., Blakeley, J.,
Lim, M., Brem, H. and Quiñones-Hinojosa, A. 2013. Multiple resections for patients with
glioblastoma: prolonging survival. Journal of Neurosurgery. 118(4), pp.812–820.
Condic-Jurkic, K., Subramanian, N., Mark, A.E. and O’Mara, M.L. 2018. The
reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in
a membrane environment. PLOS One. 13(1), p.e0191882.
Heming, C.P., de Souza Barbosa , I., Miranda, R.L., Ugarte, O.N., de São José,
V.S., Moura-Neto, V. and Aran, V. 2025. P-Glycoprotein Drives Glioblastoma Survival
and Chemotherapy Resistance. American Journal Of Pathology.
Ishibashi, K., Kezuka, Y., Kobayashi, C., Kato, M., Inoue, T., Nonaka, T.,
Ishikawa, M., Matsumura, H. and Katoh, E. 2014. Structural basis for the recognition-
evasion arms race between Tomato mosaic virus and the resistance gene Tm-1.
Proceedings of the National Academy of Sciences of the United States of America.
111(33), pp.E3486-95.
Loo, T.W. and Clarke, D.M. 2014. Tariquidar inhibits P-glycoprotein drug efflux
but activates ATPase activity by blocking transition to an open conformation. Biochemical
Pharmacology. 92(4), pp.558–566.
Lustig , S.D., Kodali , S.K., Longo, S.L., Kundu, S. and Viapiano, M.S. 2022.
Ko143 Reverses MDR in Glioblastoma via Deactivating P-Glycoprotein, Sensitizing a
Resistant Phenotype to TMZ Treatment. Anticancer Research. 42(2), pp.723–730.
Martin, C.A., Berridge, G., Mistry, P., Higgins, C.F., Charlton, P. and Callaghan,
R. 1999. The molecular interaction of the high affinity reversal agent XR9576 with P-
glycoprotein. British Journal of Pharmacology. 128(2), pp.403–411.
Mora Lagares, L., Pérez-Castillo, Y., Minovski, N. and Novič, M. 2022.
Structure–Function Relationships in the Human P-Glycoprotein (ABCB1): Insights from
Molecular Dynamics Simulations. International Journal of Molecular Sciences. 23(1),
p.362.
Reference List
Muller, Y.A., Christinger, H.W., Keyt, B.A. and de Vos, A.M. 1997. The crystal
structure of vascular endothelial growth factor (VEGF) refined to 1.93 Å resolution: multiple
copy flexibility and receptor binding. Structure. 5(10), pp.1325–1338.
Rajaratnam, V., Islam, M.M., Yang, M., Slaby, R., Ramirez, H.M. and Mirza, S.P.
2020. Glioblastoma: Pathogenesis and Current Status of Chemotherapy and Other Novel
Treatments. Cancers. 12(4).
Shapiro, A.B. and Ling, V. 1997. Positively Cooperative Sites for Drug Transport
by P‐Glycoprotein with Distinct Drug Specificities. European journal of biochemistry. 250(1),
pp.130–137.
Teodori, E., Dei, S., Bartolucci, G., Perrone, M.G., Manetti, D., Romanelli, M.N.,
Contino, M. and Colabufo, N.A. 2017. Structure-Activity Relationship Studies on 6,7-
Dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline Derivatives as Multidrug Resistance
Reversers. ChemMedChem. 12(16), pp.1369–1379.
University of Leeds 2025. Leeds Harvard referencing examples | Study and
research support | Library | University of Leeds. library.leeds.ac.uk. [Online]. Available from:
https://library.leeds.ac.uk/referencing-examples/9/leeds-harvard.
University of Leeds 2024. Literature searching explained. Leeds.ac.uk. [Online].
Available from: https://library.leeds.ac.uk/info/1404/literature-searching/14/literature-
searching-explained.
Ward, A.B., Szewczyk, P., Grimard, V., Lee, C.-W. ., Martinez, L., Doshi, R., Caya,
A., Villaluz, M., Pardon, E., Cregger, C., Swartz, D.J., Falson, P.G., Urbatsch, I.L.,
Govaerts, C., Steyaert, J. and Chang, G. 2013. Structures of P-glycoprotein reveal its
conformational flexibility and an epitope on the nucleotide-binding domain. Proceedings of
the National Academy of Sciences. 110(33), pp.13386–13391.

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