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P-glycoprotein Pamphlet: Iteration 1 of 5
- 1. P-glycoprotein Inhibition: A Novel Treatment for Glioblastoma 15 Reference List 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
- 2. 14 Conclusions and Key Take-aways 1 Contents 1. Introduction to glioblastoma………………………….2-4 1.1 What is glioblastoma? …………………………..2 1.2 Why is p-glycoprotein relevant to glioblastoma treatment?............................................................3-4 2. What is p-glycoprotein?.............................................5-7 2.1 What does p-glycoprotein look like?................5-6 2.2 How does it bind substrates?.............................7 3. Inhibiting p-glycoprotein………………………………8-11 3.1 Model of inhibition…………………………………8 3.2 Preventing ATP binding…………………………..9 3.3 Preventing substrate binding………………..10-11 4. Application to glioblastoma treatment…………….12-13 5. Conclusions and main take aways…………………….14
- 3. 13 Applications to Glioblastoma Treatment Introduction: What is p-glycoprotein? P-glycoprotein (p-gp) is a transmembrane protein which is part of the ATP binding cassette family (also described as an ABC transporter). It is predominantly found in secretory membranes such as that of the liver, ovaries and blood brain barrier (BBB). Its function is to expel toxic substances from these highly sensitive areas to prevent damage to vital organs, such as the brain (Lin and Yamazaki, 2003). In healthy human cells p-gp is vital to survival, however in cancerous cells expression of p-gp can prevent chemotherapeutic drugs from reaching the malignant cells. This presents p- gp as a pertinent drug target for preventing chemotherapy resistance in some cancers, including in glioblastoma treatment. What is glioblastoma? There are many proteins in the body that are integral to daily function, so why focus on p-gp? This protein is a significant factor in drug resistance pathways; glioblastoma is a type of cancer where this is particularly important. This is an aggressive form of brain cancer which develops from the supporting glial cells in the central nervous system (CNS) which mutate to form a solid tumour. The prognosis for glioblastoma averages at 15 months post-diagnosis and the most implemented treatment is radiotherapy followed by surgical intervention and often adjuvant chemotherapy (Rajaratnam et al., 2020). This method is far from ideal, as there are significant dangers associated with brain surgery – it has been shown that glioblastoma will likely show recurrence, and multiple resections will be necessary to improve survival. 2 Figure 1. Magnetic resonance image (MRI) of a glioblastoma (solid mass highlighted in red) in the left side of the brain. Image modified from Thakkar et al., (2024) Even then, studies have shown that only 57% of patients who underwent 4 different resection operations survived up to 24 months post- diagnosis (Chaichana et al., 2013). Similarly to Ko143,
- 4. 12 Applications to Glioblastoma Treatment CH3 CH3 Adenine Guanine CH3 N3-methyladenine N7-methylguanine O6-methylguanine Nucleic acid Nucleic acid + TMZ Interstrand crosslink Figure 2. An illustration of the alkylation (addition of CH3 groups shown in red) of nucleotides by temozolomide which results in the cross-linking of strands of DNA. Guanine can be alkylated in 2 areas: the 6th oxygen or the 7th nitrogen whereas adenine can only be alkylated at the 3rd nitrogen. The addition of alkyl groups causes carbons chains to form, resulting in cross-linking of strands of DNA which prevents proper transcription. If transcription cannot occur, proteins cannot be created resulting in the apoptosis of cells. Figure created using BioRender and Microsoft PowerPoint; informed by Lee (2016) How is P-gp Implicated in Glioblastoma? P-gp becomes relevant when discussing chemotherapeutic techniques for treating the cancer. The most common adjuvant chemotherapy is temozolomide (TMZ) an alkylating agent (Fernandes et al., 2017) which targets DNA of cancerous cells, preventing further synthesis as shown in figure 2. 3 Although there have been multiple generations of p-gp inhibitors established, none have yet been applied to standard glioblastoma (GBM) treatment due to their toxicity (Ughachukwu and Unekwe, 2012). One example of inhibitors are being applied to GBM is through the adaptation of fumitremorgin C, an inhibitor of another ABC binding cassette (breast cancer resistance protein). How does it work? • Binds to TMD • Prevents ATPase activation • Inhibits by preventing conformational change (Nielsen, 2002) Follows cycle B (figure 5) much like tariquidar Despite its effectiveness in inhibiting p-gp (and BCRP) it had high neurotoxicity when tested. As a result, an analogue, Ko143 was established – this drug showed only 1% cell death compared to a control. It was found that when temozolomide (TMZ) (chemotherapeutic agent – refer to page 3) was administered with Ko143 compared to alone, there was greater GBM cell death (Lustig et al., 2022). This is seen in figure 7. Figure 7. Bar graph comparing the administration of TMZ and Ko143 to GBM cells against TMZ alone. The bars in the highlighted box (400 and 800µM) show the greatest difference in cell viability and were statistically significantly different between the two groups (where p=0.05). Figure adapted from (Lustig et al., 2022).
- 5. 11 Inhibiting P-glycoprotein – Tetrazole and Oxadiazole Based on the research that highlighted tariquidar as a potent inhibitor, bioisosteres of this inhibitor have been more recently established. The aim of this research was to increase selectivity and improve the pharmacokinetic and pharmacodynamic profiles of the proposed drug. This was achieved by: • Alteration of the aryl amide group (non-essential to binding) • Changing hydrogen bonds in the drug binding pocket Why is an understanding of protein structure essential to tariquidar’s development? The developed compounds were found to have greater selectivity but less potency than tariquidar (Tedori et al., 2017). Building on this, Braconi et al. (2023) created bioisosteres of tariquidar (and similar compound, elacridar). The amide groups were replaced with heterocyclic rings, tetrazole and oxadiazole. Key Points So Far… So far, this brochure should have clarified: • What p-glycoprotein is • What it looks like • How can understanding p-gp’s structure inform its inhibition • There are a range of methods of inhibition • What glioblastoma is • Why it is a relevant point of research The last section of this brochure will tie all of this information together, clarifying how it can be used to create therapeutic treatments for glioblastoma. 4 Resistance to chemotherapies, including to TMZ, is multifactorial; one of the first line defences that the cancer utilises is p-gp, as it can pump TMZ out of the target cell. This means it cannot have an alkylating effect on DNA and apoptosis will not occur. How does p-gp pump out toxins? P-gp is found within the BBB as well as in the cancerous glioma cells themselves – but how does it pump out toxins? The basic principle can be defined in 5 steps (Al-Shawi and Omote, 2005) (in correlation with figure 3): 1. ATP binds to the nucleotide binding domain while TMZ (transport substrate) binds to an alternative site within the protein 2. ATP hydrolyses to ADP + pi, dephosphorylation releasing energy 3. This results in the ‘snapping’ of the protein to an open position facing the extracellular side 4. ATP binds again, while TMZ unbinds from p-gp, releasing it out of the cell 5. ATP hydrolyses, releasing energy causing the ‘snap’ back to an intracellularly open position Figure 3. Diagram depicting the transport cycle of p-glycoprotein. ATP (green diamond) shown to bind to the nucleotide binding domain (dark blue circles) causing the efflux of temozolomide (TMZ, red triangle) from the intracellular to extracellular side of the plasma membrane. Figure created using Microsoft PowerPoint.
- 6. 10 Inhibiting P-glycoprotein – Tariquidar There have been a number of p-gp inhibitors that follow pathway B (seen in figure 5), one of the most promising being tariquidar. This drug stops cycling by holding p-gp in the closed formation (shown in figure 6), preventing cycling (Loo and Clarke, 2014). Figure 6 shows the two NBDs as being held together in a dimerized formation in the ‘closed’ conformation. This means that ATP hydrolysis can still be activated, yet there is no snapping to the open conformation. Certainty surrounding binding to the TMD rather than the NBD was clarified because inhibition still occurred in the absence of NBDs (mutated version of p-gp). Figure 6. 3D illustration of the open and closed conformations of p-glycoprotein; consideration to be shown to the dimerization of the NBDs in the closed conformation. Figure taken from Loo and Clarke (2014) created using Pymol. Although it is not known exactly where it binds, it is thought that the site is close to areas in the TMDs that are near extracellular loops 1 and 4. The movement of these loops determine the cross linking of residues that hold the protein open. When bound, tariquidar prevents the cross linking of these residues, therefore stopping p-gp from being held in the open conformation cycling (Loo and Clarke, 2014). However, tariquidar did not pass clinical trials because of toxicity, adverse effects and unwanted pharmacokinetic interactions (Tedori at al., 2017). Despite this, it did create a basis for the further development of inhibitors that work using a similar mechanism. How does tariquidar hold p-gp closed? 5 Based on an understanding of p-gp’s function, and how its structure informs its function, therapies to inhibit the process shown in figure 3 could allow vast improvements in glioblastoma chemotherapy. Inhibition of p-gp would remove a first line defence of glioblastoma, preventing multi-drug resistance (MDR) and reducing the chance of recurrence. What does p-gp look like? To inhibit this protein, it is integral that its structure is understood, as the structure of p-gp directly informs its function. Although the basic transport cycle of p-gp is well established, it is a complex quaternary protein, made up of two halves that both contain a transmembrane domain and nucleotide binding domain as illustrated in figure 4. These domains have distinct structures and therefore differing functions. Transmembrane Domains • Within the membrane • 6 hydrophobic alpha helices • Variable amino acid sequences • Move between the inward and outward facing positions Nucleotide Binding Domains • Within the cytosol • Highly conserved comprised of Walker A, B and signature motifs • Flexible structure allows polyspecificity • Where ATP is hydrolysed The domains are connected by intracellular loops (ICLs) known as linker regions. When the two nucleotide binding domains dimerise, this forms the ATP binding site (Mora Lagares et al., 2022). Figure 4. An illustration of the crystal structure of p- gp. TMDs are depicted in cyan and yellow; NBDs are shown in magenta. Figure taken from Mora Lagares et al., (2022)
- 7. 9 Inhibiting P-glycoprotein: Preventing ATP Binding Following model A (illustrated in figure 5), there is evidence to suggest that inhibition of ATP binding at the NBD can prevent p-gp function. Ward et al. (2013) created overlapping crystal structures of p-gp; the binding of the nanobody Nb592 inhibits ATP binding by acting as a competitive inhibitor. Depicted in figure 6, there were 2 models produced by this study showing Nb592 bound to the c-terminal of NBD1 (shown in red in the figure). Attachment here prevents the dimerization of the two nucleotide binding domains – this conformational change is essential to the binding of ATP. Its hydrolysis (and release of a phosphate) is what initiates the snapping mechanism, characteristic of p-gp. Figure 6. Illustration of the crystal structure of p-gp with Nb592 bound. A shows the full crystal structure, including the transmembrane domains (labelled TMD1/2) and the NBDs (1/2). Nb592 (red) is bound to NBD1 which will prevent dimerization of the two NBD. B shows a more detailed attachment of Nb592 binding at NBD1, where complimentary determining region (CDR) 3 (green) fits into the pocket created by three alpha helices. The Walker A motif (discussed on page 5) is shown in cyan. Figure taken from Ward at al. 2013). This study created a potential molecular scaffold for an inhibitor of p-glycoprotein based on a structural understanding of the protein. Forcing p-gp to remain in a stabilised, closed conformation shows a promising method for inhibiting this protein’s function, and in turn, preventing MDR in glioblastoma. Motifs are sequences of amino acids that are highly conserved and correlate to specific protein function. Walker A: Codes for the phosphate binding loop (p-loop) that allows ATP binding (Oiwa and Sakakibara, 2005). Walker B: This sequence interacts with Mg2+ allowing conformation of the ATP binding site (Ramaen et al., 2006). Signature (ABC): This combination of amino acids senses ATP ϒ-phosphate binding ensures conformational change when ATP is hydrolysed (Hewitt and Lehner, 2003). Understanding the primary structures that make up these motifs gives a direct indication of function. 6 After exposure to multiple different agonists, there were the greatest number of conformational changes to the NBDs and loop regions. One extracellular loop (residues 90-100) was thought to be significant in preventing substances from re- entering the cell, by inducing a rapid closure of the protein on the extracellular side (Mora Lagares et al., 2022). How could this be a useful therapeutic target for glioblastoma treatment? Nucleotide Binding Site Motifs
- 8. 8 Inhibiting P-glycoprotein Understanding what p-gp looks like can inform how one can aim to inhibit it; the development of crystal structures (as aforementioned) allows the presentation of 2 methods of inhibition (as shown in figure 5). Inhibiting the transport binding sites would prevent the efflux of TMZ as it would not be able to bind to p-gp before the conformational change. Preventing ATP binding results in the protein remaining unhydrolyzed, preventing the conformational change that determines efflux. TMZ Figure 5. Diagram depicting 2 potential target areas to inhibit p-glycoprotein’s function. A (brown arrows)shows inhibition at the NBD (dark blue circles) where ATP (green diamond) would normally be hydrolysed; an inhibitor (red diamond) prevents binding and therefore the conformational change that would cause efflux of TMZ (purple triangle). B (red arrows) illustrates inhibition due to an inhibitor (red trapezium) a transport binding site at the TMD. 7 Substrate Binding As well as having to binds ATP to the NBDs to activate conformational change, the substrate being transported across the membrane must bind to p- gp (e.g., TMZ as in figure 3). Due to the polyspecific nature of this protein, there are many unknowns surrounding how it manages to bind such a variance of substrates. Since the 1990s it has been known that there are multiple substrate binding sites on p-gp. One of the first proven instances of this was shown by Shapiro and Ling (1997) who established that the dyes rhodamine 123 and Hoechst 33342 could bind simultaneously to p-gp. The main takeaways included: • The coining of the ‘H’ and ‘R’ sites in the transmembrane domains • Simultaneous binding has a positively cooperative effect Further research by Martin et al., (1999) illustrated that the p-gp transport substrates vinblastine and paclitaxel showed 4- and 20- fold lower affinity, respectively, when the substrate XR9576 was bound. This developed the idea that there could be 2 classes of binding sites: 1. Transport binding sites 2. Regulatory binding sites Since 2000, more and more sites are being discovered, such as the progesterone binding site (‘p’ site) (Shapiro et al. 2001). Some of these sites are solely transport or regulatory sites, and others are thought to have properties of both (Martin et al., 2000). How could this inform a potential method of inhibiting of p- glycoprotein?