P-glycoprotein Pamphlet: Iteration 2 of 5

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
1. How to use guide……………………………………...2-3
2. Introduction…………………………………………….4-5
1.1 Introduction to glioblastoma.
…………………..4
1.2 How is P-glycoprotein implicated in
glioblastoma?........................................................5
3. What is P-glycoprotein?.............................................6-7
2.1 What does P-glycoprotein look
like?..................6
2.2 How does P-gp bind
substrates.........................7
4. Inhibiting P-glycoprotein………………………………8-11
3.1 Model of
inhibition…………………………………8
3.2 Examples of P-gp
inhibition…………………….9-11
5. Application to glioblastoma treatment…………….12-13
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, (chapters 2-5 as laid
out in the contents page) 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.
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 11) 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.
Page 3 shows (with images) how to access the podcast
recordings.
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.

4
Introduction to Glioblastoma
P-glycoprotein (P-gp) is a transmembrane protein relevant to the progression
of glioblastoma, a type of solid brain tumour. There are many types of cancer
that require improvement in treatment, so why focus on glioblastoma (GBM)?
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.
Use the abstract and introduction in this paper to answer:
1. How common is glioblastoma is 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?
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.
(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).
(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.
5
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:
5. What P-gp is and how its expression is altered in glioblastoma.
6. How its expression affects the delivery of chemotherapeutic drugs to a
tumour.
i. How the delivery of chemotherapeutic drugs might be
altered if P-gp is removed (knocked down).
(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.

What Does P-glycoprotein 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-glycoprotein
using cryo-electro magnetic imaging.
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).
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.
(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.
How Does P-gp Bind Substrates?
As discussed in paper 4, P-glycoprotein 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.
(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.
(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.
7
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 (focus on pages 131-133) paper 5,
describe:
11. The main conclusions drawn from the paper regarding the binding sites
of P-gp.
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.
Figure 2. 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
6

Model 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-glycoprotein.
Using the introduction and figure 1a from paper 7, as well as your answers
to questions 7-12, fill in the diagram below.
14. Describe and explain the process of inhibition you can see in:
i. Cycle A
ii. Cycle B
15. Why is it integral that the structure of P-gp is understood to suggest the
methods of inhibition shown above?
8
P-gp Inhibition: Nb592
(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.
9
Based on the models shown on page 8, 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:
16. 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).
17. Explain where Nb592 binds to P-gp.
18. Which path of inhibition does Nb592 binding initiate? Refer to the diagram
on page 8.
19. Explain how Nb592 causes the selected inhibition pathway.
(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 3. 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)
B C

10 11
P-gp Inhibition: Tariquidar
One of the more promising inhibitors of P-gp that has been studied in vitro is
tariquidar. This 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 8)?
20. How is P-gp held on a closed conformation? (Consider where drug binding
occurs).
21. How does holding P-gp closed stop its transport cycle?
(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.
(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.
Despite tariquidar showing promising inhibition of P-gp in vitro, in vivo studies
did no 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’.
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.
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 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.
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/140
4/literature-searching/14/literatur
e-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/referen
cing-examples/9/leeds-harvard
.

12 13
Application to Glioblastoma Treatment: Ko143
It has now been explored how attempts to inhibit P-gp - based on an
understanding of its structure - have been made, 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?
(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.
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 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! Have a go at
looking at the 3D structures of proteins using their PDB ID;
access BioRender here: https://www.biorender.com/

14 15
Conclusions and Key Take-aways Reference List
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.
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.
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.
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

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.
16

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