Antibiotic resistance is the overarching process whereby bacteria become resistant to antibiotics (medication that would normally kill them). The issue can result in a number of antibiotics no longer being effective when taken by a patient to fight an infection. Due to this, many serious health issues can be triggered since your body will have to fight off the infection without the support of antibiotics, which results in a longer treatment period, potentially worse symptoms and side effects, and could even result in death if the body's immune system cannot fight off the infection sufficiently.

How does antibiotic resistance actually come about?

There are a number of different mechanisms by which bacteria can develop resistance to antibiotics. I shall outline three key examples in detail, and then provide some other methods which you can look into further if you are interested!

1. Horizontal Gene Transfer (HGT): the sharing of genes between bacteria. There are three mechanisms of HGT: transformation, transduction and conjugation. Transformation occurs when bacteria take up DNA from its surrounding environment and integrate it into their overall genome. Transduction is a process whereby bacteriophage viruses inject viral DNA into bacteria. Finally, conjugation occurs when two bacterial cells engage in contact leading to the transmission of a plasmid (small circular loop of DNA) from one cell to the other through a cell membrane structure found in bacteria called the sex pili.
2. Target Site Modification: when the target site of an antibiotic on the bacterium is modified. This is a very common mechanism that bacteria use to develop resistance to antibiotics. When bacteria copy their genome during replication, mistakes in the DNA sequence can occur. If any mutation(s) occur in a gene that encodes for a protein which is the target of an antibiotic, the antibiotic may no longer be able to bind to the target. Those with the mutation thus have a selective advantage, and reproduce, causing the mutation to spread further throughout the population as it grows.
3. Efflux Pumps: obtaining additional efflux pumps as a result of the alteration of proteins that control the amount of efflux pumps produced, and hence chromosomal mutations can increase resistance. Efflux pumps pump a specific type of antibiotic outside of the bacterial cell, hence, lowering the intracellular antibiotic concentration. The overproduction of efflux pumps thus leads to an increased resistance to the drug targeted by the pumps.
4. Intrinsic Resistance: where a bacterial species is naturally resistant to a certain antibiotic/family of antibiotics without the need for mutation or mutation of further genes - the 2 major mechanisms by which bacteria mediate intrinsic resistance are by differences in membrane permeability and access.
5. Enzyme Inactivation
6. Enzyme Modification: modify the target of the antibiotic or the antibiotic itself
7. Replacement of the Target Site
8. Overproduction of the Target
9. Transposons: autonomous mobile genetic elements typically found integrated in the host chromosome; contain genes that enable them to integrate and excise from the chromosome
10. Mixed Mechanisms

With the current situation revealing bacterial species that are resistant to all or almost all available antibiotics, the crisis is becoming more and more concerning. One key example of this are carbapenem-resistant enterobacteriaceae (CRE), which have become resistant to all/nearly all currently available antibiotics, including carbapenems. Carbapenems are a class of antibiotics typically reserved for final use/last resort, so when bacteria become resistant to these too, the situation's worrying nature deepens significantly.

Global Perspectives

A few key points to consider when evaluating the crisis relating to antimicrobial resistance:

• We, as citizens of Earth, need to work together as a team to tackle this crisis - investing money into it and ensuring that every country, and person, has equal access to antibiotics where they are needed, but also to the medical expertise to inform whether or not antibiotics are actually required for a certain condition
• Usage is a big issue - farmers are known to use a lot of antibiotics, but to what extent is unknown (they are used for prophylaxis, treatment, etc.). Recently, through the Ministry of Livestock, Agriculture and Fisheries (UK), the addition of antimicrobials to food for animal feeds has been almost entirely outlawed
• More antimicrobial resistance occurs in cities compared with rural sites due to more readily available access to antimicrobials.
• Buying antibiotics over the counter without prescription is illegal (UK) - laws are there against that are we are unable to effectively carry out mandates in order to enforce them. There is a lot of misuse and abuse of antibiotics in that context.
• We do not always have enough data to convince policy makers regarding the big issue of antimicrobial resistance - for example in the UK, there is currently no national surveillance programme to collect data consistently.
• Kenya's government adopted the National Action Plan that emanated from the Global Action Plan - if implemented fully, the five main objectives will ensure that we are able to start to tackle antimicrobial resistance there.
• New technologies to perform surveillance faster and gather data much faster on usage in both humans and livestock must be developed. We also need the facilities to collect data on how many antibiotics are used in hospital, in the community, and more importantly for what purpose as well as how much is misused.

Some key take home messages from this relatively short article:

• Ensure to finish your entire course of antibiotics if you are prescribed them, regardless of whether you feel better or not after say half of your course. Stopping the course part way through could lead to the bacteria being able to develop more resistance against that antibiotic.
• Only take antibiotics when they are prescribed to you by a licensed medical professional and hence issued by a pharmacist. If you believe that you may not require the antibiotics, remember that it is always okay to ask questions. If you do truly need them, the medical staff will be able to explain why and put your mind at ease.
• Keep spreading the message of how serious this issue is - hopefully with lots of media attention, the issue may be addressed more frequently around the globe in order to reduce the issue before we see the issue spiral completely out of control. Take part in WAAW (detailed below) at your school/university/work place! Find a link to the WAAW page in the references!

Remember! World Antimicrobial Awareness Week (WAAW) 2021: 18th November - 24th November

5 important goals of WAAW as outlined by the WHO:

1. Raising awareness about antibiotics
2. Increasing research and supervision
3. Reducing infections
4. Using antimicrobial medications the right way
5. Committing to continual investment

“Antibiotic resistance occurs when bacteria change in response to the use of these medicines. Bacteria, not humans or animals, become antibiotic-resistant.” - WHO

I hope this article was useful and an interesting read - check back tomorrow for yet another instalment on biochemistree! Up next: "Are Viruses Alive?"

References

Antibiotic Research UK  About Antibiotic Resistance [online] Available At: https://www.antibioticresearch.org.uk/about-antibiotic-resistance/ Date Last Accessed: 05/12/2020
Lambert, P.A. (2005) Bacterial resistance to antibiotics: Modified target sites Advanced Drug Delivery Reviews doi:
10.1016/j.addr.2005.04.003
von Wintersdorff C.J.H., Penders J., van Niekerk, J.M., Mills, N.D., Majumder, S., van Alphen, L.B., Savelkoul, P.H.M. and Wolffs , P.F.G. (2016)
Dissemination of Antimicrobial Resistance in Microbial Ecosystems through Horizontal Gene Transfer Frontiers in Microbiology doi:
10.3389/fmicb.2016.00173
Webber, M.A. and Piddock, L.J.V. (2003)
The importance of efflux pumps in bacterial antibiotic resistance Journal of Antimicrobial
Chemotherapy doi: 10.1093/ jac /dkg050
Wellcome Genome Campus Advanced Courses and Scientific Conferences
Mechanisms of resistance Retrieved From FutureLearn:
https://www.futurelearn.com/courses/introduction-to-bacterial-genomics/6/steps/831670 Date Last Accessed: 05/12/2020
Wellcome Genome Campus Advanced Courses and Scientific Conferences
Resistance through chromosomal mutation Retrieved From FutureLearn: https://www.futurelearn.com/courses/introduction-to-bacterial-genomics/6/steps/831671 Date Last Accessed: 05/12/2020
Wellcome Genome Campus Advanced Courses and Scientific Conferences
Resistance through horizontal gene transfer Retrieved From FutureLearn: https://www.futurelearn.com/courses/introduction to-bacterial-genomics/6/steps/831672 Date Last Accessed: 05/12/2020

Who.int. 2021. World Antimicrobial Awareness Week. [online] Available at: https://www.who.int/campaigns/world-antimicrobial-awareness-week Date Last Accessed: 17/09/2021

A caspase is an aspartate-specific cysteine protease. To break that down a little, aspartate and cysteine are both amino acids. Caspases are named in this way due to their highly specific cysteine protease activity - a cysteine residue found within the protease's active site induces nucleophilic attack and hence cleaves a target protein, at the C-terminal of an aspartic acid residue within the protein. In effect, this cleavage of the cysteine protease triggers apoptosis through various methods, all characteristic of apoptosis, including:

• DNA fragmentation (splitting of DNA into smaller sections of DNA cutting at various points)
• externalisation of phosphatidylserine on the membrane (moving the phosphatidylserine from the interior of the cell membrane (intracellular) to the exterior of the cell membrane (extracellular))
• formation of apoptotic bodies (small membrane bound fragments that are removed by phagocytosis (engulfed by phagocytes (white blood cells)) without triggering an inflammatory response)

If a tumour causes downregulation of caspase-8, this can lead to tumour progression via the secretion of proteins into the tumour microenvironment that aid the development of the tumour according to a recent study by Kostova et al., 2020. If this is the case, then a drug which induces caspase-8 expression could be beneficial to patients. It has been shown previously that failure to regulate the expression of caspase-8 can lead to an imbalance between apoptosis and non-apoptosis, and this occurs both in the tumour, and the TME.

What is downregulation? Downregulation is ultimately a process whereby a cell decreases the quantity of a cellular component, for example a protein such as caspase-8, or RNA, in response to an external stimulus.

The tumour microenvironment contains a multitude of cytokines - proteins which are secreted by cells that can fall into various different categories including one key category in this case, interleukins. Interleukins are produced by a leukocyte and lead to an effect on another leukocyte. CCL2 and IL-6 are both examples of cytokines which can have their effects mimicked by other leukocytes known as macrophages, which have been polarised.

This polarisation can occur as a result of manipulation by caspase-8 inhibitors. The polarisation of macrophages using caspase-8 to give M1 and/or M2 macrophages has already been evidenced through a mouse model. The model exemplified that the polarisation was required in order for the differentiation of monocytes to occur. Furthermore, in relation to the previous article covering trabectedin and lurbinectedin (targeting the tumour microenvironment), both of these drugs have been shown to reduce the production of growth factors including the aforementioned CCL2 and IL-6. Moreover, this in term leads to changes in the cytokine expression found within the tumour microenvironment, as well as their typical function (which is inhibiting the growth of the primary tumour). Since it is know that caspase-8 can also interact with the growth factors, the suggestion that caspase-8 could play a significant role in communicating between the tumour and its microenvironment is further strengthened. In addition to this, caspase-8 also regulates angiogenesis, proposed by some to use an interleukin mediated pathway. As a result of this, drugs including trabectedin and lurbinectedin could therefore access the regulation of angiogenesis and cytokine expression through the regulation of caspase-8. Hence, their targeted therapeutic nature by which they can target the tumour's microenvironment could be explained.

The entire role of caspase-8 is currently unclear. The points below outline a few current thoughts:

• Current research makes it apparent that caspase-8 could mediate whether the environment is pro-tumourigenic or anti-tumourigenic through the means of controlling how lymphocytes differentiate.
• The investigation of potential signalling pathways between caspase-8, tumour cells (especially active ones) and the tumour microenvironment could aid a development in understanding how the tumour microenvironment becomes pro-tumourigenic.
• Is caspase-8 relevant in mechanisms behind how an anti-tumour environment is maintained in non-tumour cells?
• Do mutations interact with caspase-8 in any way? Or can mutations interact with the way that caspase-8 communicates between cells and their environments?

Thoughts to consider!

• The TME contains numerous components which can regulate the development and progression of cancerous tumours
• So far, developments in understanding the TME have led to the development of targeted therapeutics including trabectedin and lurbinectedin which target both the tumour cells, and the TME
• The role of caspase-8 in both apoptosis and the regulation of TME composition demonstrates potential for caspase-8 to act as a link between the tumour itself and the TME
• Advancements in the area surrounding caspase-8 could lead to the development of further targeted therapeutics which in turn could improve patient outlooks in many ways

Can you think of any more potential links that caspase-8 could have a role in? To help, I will give you four main broad categories to consider (all of which have been touched on at points in this article, but could be explored in more depth!):

• Regulation
• Therapeutic Targets
• Controlling Cell Differentiation
• Signalling

For example: a couple of additional points!

• IL-6 causes monocytes to tend to differentiate into macrophages instead of dendritic cells. What are the potential effects of causing further differentiation to be favoured towards one type of cell? What advantages and disadvantages could that have?
• Is it unique to this case? Could some of the other caspases which work in apoptosis or the immune response lead to any similar results? For reference (note how caspase-8 is the only one to appear in both an apoptotic and immune response category):
• caspases -2, -8, -9 and -10 are apoptotic initiators
• caspases -3, -6, and -7 are apoptotic effectors
• caspases -1, -4, -5, -8 and -12 are related to immune response

I hope that this relatively short article has provided a few thinking points to open your mind to the sort of questions that can be asked in this area as a result of current studies and what we would like to know to advance next! Also, I hope that you are all looking forward to the upcoming articles now that I've finished a busy summer including jury service and much more! Lots of articles scheduled to be released this week! Looking forward to hearing from you all.

References

Antonopoulos, C. and Dubyak, G. (2014) Chemotherapy engages multiple pathways leading to IL-1β production by myeloid leukocytes OncoImmunology doi: 10.4161/onci.27499

Belgiovine, C., Bello, E., Liguori, M., Craparotta, I., Mannarino, L., Paracchini, L., Beltrame, L., Marchini, S., Galmarini, C. M., Mantovani, A., Frapolli, R., Allavena, P. and D’Incalci, M. (2017) Lurbinectedin reduces tumour-associated macrophages and the inflammatory tumour microenvironment in preclinical models British Journal of Cancer doi: 10.1038/bjc.2017.205

Budd, R.C. (2002) Death receptors couple to both cell proliferation and apoptosisThe Journal of Clinical Investigation doi: 10.1172/JCI15077

Chomarat, P., Banchereau, J., Davoust, J. and Palucka, A.K. (2000) IL-6 switches the differentiation of monocytes from dendritic cells to macrophages Nature Immunology doi: 10.1038/82763

Cuda, C.M., Misharin, A.V., Khare, S., Saber, R., Tsai, F., Archer, A.M., Homan, P.J., Kenneth Haines III, G., Hutcheson, J., Dorfleutner, A., Budinger, G.R.S., Stehlik, C. and Perlman, H. (2015) Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner Arthritis Research & Therapy doi: 10.1186/s13075-015-0794-z

Ferraro-Peyret, C., Quemeneur, L., Flacher, M., Revillard, J-P. and Genestier, L. (2002) Caspase-independent phosphatidylserine exposure during apoptosis of primary T lymphocytes Journal of Immunology doi: 10.4049/jimmunol.169.9.4805

Germano, G., Frapolli, R., Simone, M., Tavecchio, M., Erba, E., Pesce, S., Pasqualini, F., Grosso, F., Sanflippo, R., Casali, P.G., Gronchi, A., Virdis, E., Tarantino, E., Pilotti, S., Greco, A., Nebuloni, M., Galmarini, C.M., Tercero, J.C., Mantovani, A., D’INcalci, M. and Allavena, P. (2010) Antitumor and Anti-inflammatory Effects of Trabectedin on Human Myxoid Liposarcoma Cells Cancer Research doi: 10.1158/0008-5472.CAN-09-2335

Kerr, J. F. R., Wyllie, A. H. and Currie, A. R. (1972) Apoptosis: A Basis Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics British Journal of Cancer doi: 10.1038/bjc.1972.33

Kostova, I., Mandal, R., Becker, S. and Strebhardt, K. (2020) The role of caspase-8 in the tumor microenvironment of ovarian cancer Cancer and Metastasis Reviews doi: 10.1007/s10555-020-09935-1

Larsen, A.K., Galmarini, C.M. and D’Incalci, M. (2016) Unique features of trabectedin mechanism of action Cancer Chemotherapy and Pharmacology doi: 10.1007/s00280-015-2918-1

Roca, H., Varsos, Z.S., Sud, S>, Craig, M.J., Ying, C. and Pienta, K.J. (2009) CCL2 and Interleukin-6 Promote Survival of Human CD11b⁺ Peripheral Blood Mononuclear Cells and Induce M2-type Macrophage Polarization Journal of Biological Chemistry doi: 10.1074/jbc.M109.042671

Salmena, L., Lemmers, B., Hakem, A., Matysiak-Zablocki, E., Murakami, K., Au, B.Y.B., Berry, D.M., Tamblyn, L., Shehabeldin, A., Migon, E., Wakeham, A., Bouchard, D., Yeh, W.C., McGlade, J.C., Ohashi, P.S. and Haken, R. (2003) Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity Genes & Development doi: 10.1101/gad.1063703

Tummers, B. and Green, D. R. (2017) Caspase-8; regulating life and death Immunological Reviews doi: 10.1111/imr.12541

Zhang, J-M. and An, J. (2007) Cytokines, Inflammation and Pain International Anaesthesiology Clinics doi: 10.1097/AIA.0b013e318034194e

The aims of modern platelet research are balanced between the prevention of heart attack and stroke through the inhibition of platelet activation, while ensuring that platelets are not inhibited entirely in order to prevent bleeding complications for the patient.

Current Treatments

Standard treatments for arterial thrombosis currently use aspirin and clopidogrel. Prasugrel is also being used instead of clopidogrel to block the P2Y12 G-protein coupled receptor (GPCR) which is proving to be more effective at reducing the risk of stroke and heart attack. However, it does carry an increased bleeding risk compared with clopidogrel. Contrarily, aspirin targets a component of the thromboxane pathway called cyclooxygenase. Other drugs targeted to inhibit the αIIbβ3 integrin (such as eptifibatide and abciximab) have also been developed - these are seen to be used during stenting procedures to open up blood vessels where restriction through means of an atherosclerotic plaque or otherwise has occurred. Though, as a result of high bleeding risk associated with their use, they are not used for long term treatments. Anticoagulants can suppress platelet activation due to their ability to directly inhibit thrombin, and hence some of these can also be used in the treatment of arterial thrombosis.

So, what are these components? What are we actually targeting?

• P2Y12 G-protein coupled receptor (GPCR): a GPCR is a cell surface receptor which acts to detect messages through signalling molecules such as peptides, light energy, lipids and sugars to inform the cell about its environment. They consist of a single globular polypeptide, with seven segments that span the entire width of the membrane, with intervening portions looping inside and outside of the cell (shown in Figure 1). GPCRs interact with G proteins in the plasma membrane. When a signalling molecule binds the GPCR, a conformational change is induced, thus triggering an interaction between the GPCR and a nearby G protein. P2Y12 is a chemoreceptor (chemical receptor) for adenosine diphosphate (ADP).
• Thromboxane A2 (TXA2; GPCR): also a GPCR. TXA2 is a potent stimulator of platelet aggregation, and its activity is mediated through a G protein that activates a phosphatidylinositol-calcium second messenger system. Aspirin targets this particular receptor.
• αIIbβ3 integrin: integrins are transmembrane glycoprotein (proteins modified through the addition of lipid) which are able to transmit information both ways across a plasma membrane (bidirectional transfer). αIIbβ3 is expressed at high levels in platelets are progenitors of platelets, playing a key role in platelet function, haemostasis and arterial thrombosis. It also participates in cancer progression through tumour cell proliferation amongst other systems. In resting platelets it has an inactive conformation, but upon stimulation through agonists, the transduction of signals leads to a switch to a high affinity state particularly for fibrinogen. Overall this causes integrin clustering and hence drives aggregation, thrombus consolidation and more. Hence drugs to target this integrin are produced.
• von Willebrand Factor (vWF): vWF is an adhesive and multimeric glycoprotein necessary for normal haemostasis. It has central role in many processes including the mediation of platelet adhesion to vascular sub-endothelium, and therefore also platelet aggregation. Hence, drugs to target vWF and its respective integrin (shown in Figure 1) are in development through  clinical trials.
• Collagen: appears in a platelet aggregation pathway, calcium dependent. An increase in collagen leads to an increase in the concentration of calcium present in the platelets, and hence an increase in platelet aggregation. The increase in platelet aggregation tends to be seen as a result of calcium influx from the sodium-calcium exchanger functioning in a reverse mode from the extracellular milieu. Hence, drugs are in development and trials for targeting this too.

As shown above in Figure 1, antiplatelet therapy often refers to a dual therapy combining aspirin and a P2Y12 GPCR inhibitor (clopidogrel/prasugrel) which in turn targets the activation of platelets via thromboxane and ADP. These dual therapies significantly reduce the risk of cardiovascular events compared to monotherapy or lack of treatment, but the elevated risk of major bleeding is something to also be considered. The bleeding risk is also dependent on other factors including the duration of therapy and dosage(s) prescribed.

Modern Approaches

As a result of the high bleeding risks associated with the current primary treatments, the search for novel antiplatelet drugs that have a high efficacy with respect to the treatment of arterial thrombosis, whilst having a lower (or better still, if possible, nil) bleeding risk is a key aim of current research involving platelets.

vWF, and its integrin GPIbα-GPIX-GPV (and the overall pathway associated with adhesion). vWF has been shown to be important in platelet adhesion and aggregation whereby the GPIb-vWF complex contributes to the tethering and rolling of the platelet to the vessel. This is outlined in Figure 2.  People who are deficient in vWF have von Willebrand disease bleeding disorder. Antiplatelet drugs made to target this factor and integrin will enable the risk of thrombosis to be decreased as a result of preventing platelet tethering. However, theoretically this could increase the risk of bleeding shown by vWF-deficient humans.

However, it has been found in early stage (phase I) clinical trials (Cataland et al., 2012) that one particular drug, ARC1779, shows improvements in clinical symptoms - particularly the reduction of cardiovascular events. However, as of yet no issues have arisen with respect to patient safety, including bleeding. Hence, there is the possibility that drugs such as ARC1779, or those that target in similar ways could lead to a reduction in bleeding risks for patients. Despite this, these are early stage trials and further clinical trials on larger scales with many more patients will be required in order to see whether they could be of significant benefit when used as an anti-platelet therapy since phase I trials only evaluate the efficacy of a drug within a small patient cohort, so the effects on a large population cannot yet be evaluated - including the possibility of  significant adverse events that may occur in 1 in 50 patients for example. Other drugs that target vWF, or collagen will also have to go through the safe processes. At the moment, treatments focus on these factors found within the membranes of platelets and hence there are a limited number of factors to observe and target. Regardless, it may be possible to find something which significantly reduces bleeding risk whilst still being effective, or there may be other ways which have not yet been considered such as further back in signalling networks and targeting the signalling molecules themselves that are attracted to the receptors.

Nonetheless, this progress is promising; showing that it may be possible to produce a drug with a significantly reduced risk of bleeding, even if the bleeding still occurs somewhat, especially when compared with the current treatments available. Furthermore other actions can be considered, such as if a drug is highly potent, using a lower concentration or dosage which may also decrease the risk of bleeding for the patient. Although, this cannot afford to compromise the efficacy of the drug since in many cases a lower concentration may not actually prevent thrombosis, or it could lead to a patient developing resistance to the drug before it has been able to treat the condition for a significant period of time in more circumstances due to exposure at lower dosages. However, if the drug is highly potent, it could therefore still be possible.

Conclusion

In conclusion, there are signs (as evidenced above) that antiplatelet therapy whereby less bleeding is experienced, at least in a majority of patients, may be possible to be developed. However, eliminating the risk of bleeding in its entirety seems rather unlikely at the moment. A number of factors that are the subject of new developments and possible future treatments, including vWF, are essential in the adhesion and aggregation of platelets to prevent bleeding disorders. As a result, the targeting of these in any way could lead to disruption of these normal processes such as seen through thromboxane inhibitors leading to increased bleeding risks. Drugs such as ARC1779 show potential with further study, especially if the drugs can be administered at low dosages due to high potency. Another possibility would be the drug being able to treat arterial thrombosis without triggering a vWF deficiency and hence reducing bleeding risk.

I hope that through this article I've been able to outline some of the key ideas related to treating arterial thrombosis using antiplatelet drugs - including a focus on trying to reduce the risk of bleeding disorders if possible. If you are interested in this area, feel free to ask questions in the "Any Questions?" page and I will reply as soon as possible! Upcoming article topics include caspase 8, and antimicrobial resistance, and will be far more frequent now that my exams are over. Stay tuned for more!

References

Algahtani, F. H. and Stuckey, R. (2019) ‘High factor VIII levels and arterial thrombosis: illustrative case and literature review’, Therapeutic Advances in Hematology, 10, p. 204062071988668. doi: 10.1177/2040620719886685.

Cataland, S. R. et al. (2012) ‘Initial experience from a double-blind, placebo-controlled, clinical outcome study of ARC1779 in patients with thrombotic thrombocytopenic purpura’, American Journal of Hematology, 87(4), pp. 430–432. doi: 10.1002/ajh.23106.

Eikelboom, J. W. et al.(2005) ‘Enhanced antiplatelet effect of clopidogrel in patients whose platelets are least inhibited by aspirin: a randomized crossover trial’, Journal of Thrombosis and Haemostasis, 3(12), pp. 2649–2655. doi: 10.1111/j.1538-7836.2005.01640.x.

Fan, H. et al. (2019) ‘Structural basis for ligand recognition of the human thromboxane A2 receptor’, Nature Chemical Biology, 15(1), pp. 27–33. doi: 10.1038/s41589-018-0170-9.

Ferraris, V., Ferraris, S. and Saha, S. (2011) ‘Antiplatelet Drugs: Mechanisms and Risks of Bleeding Following Cardiac Operations’, International Journal of Angiology, 20(01), pp. 001–018. doi: 10.1055/s-0031-1272544.

Fontana, P. et al. (2014) ‘Antiplatelet Therapy: Targeting the TxA2 Pathway’, Journal of Cardiovascular Translational Research, 7(1), pp. 29–38. doi: 10.1007/s12265-013-9529-1.

Gimbel, M. et al. (2020) ‘Clopidogrel versus ticagrelor or prasugrel in patients aged 70 years or older with non-ST-elevation acute coronary syndrome (POPular AGE): the randomised, open-label, non-inferiority trial’, The Lancet, 395(10233), pp. 1374–1381. doi: 10.1016/S0140-6736(20)30325-1.

Gurbel, P. A., Kuliopulos, A. and Tantry, U. S. (2015) ‘G-Protein–Coupled Receptors Signaling Pathways in New Antiplatelet Drug Development’, Arteriosclerosis, Thrombosis, and Vascular Biology, 35(3), pp. 500–512. doi: 10.1161/ATVBAHA.114.303412.

Hao, Q. et al. (2018) ‘Clopidogrel plus aspirin versus aspirin alone for acute minor ischaemic stroke or high risk transient ischaemic attack: systematic review and meta-analysis’, BMJ, p. k5108. doi: 10.1136/bmj.k5108.

Huang, J. et al. (2019) ‘Platelet integrin αIIbβ3: signal transduction, regulation, and its therapeutic targeting’, Journal of Hematology & Oncology, 12(1), p. 26. doi: 10.1186/s13045-019-0709-6.

Leebeek, F. W. G. (2019) ‘A prothrombotic von Willebrand factor variant’, Blood, 133(4), pp. 288–289. doi: 10.1182/blood-2018-11-883488.

Peyvandi, F., Garagiola, I. and Baronciani, L. (2011) ‘Role of von Willebrand factor in the haemostasis’, Blood Transfusion, pp. s3–s8. doi: 10.2450/2011.002S.

Rana, A. et al. (2019) ‘Shear-Dependent Platelet Aggregation: Mechanisms and Therapeutic Opportunities’, Frontiers in Cardiovascular Medicine, 6, p. 141. doi: 10.3389/fcvm.2019.00141.

Roberts, D. E., McNicol, A. and Bose, R. (2004) ‘Mechanism of Collagen Activation in Human Platelets’, Journal of Biological Chemistry, 279(19), pp. 19421–19430. doi: 10.1074/jbc.M308864200.

Rosenbaum, D. M., Rasmussen, S. G. F. and Kobilka, B. K. (2009) ‘The structure and function of G-protein-coupled receptors’, Nature, 459(7245), pp. 356–363. doi: 10.1038/nature08144.

Cancer is a group of over 100 diseases that involve abnormal, uncontrolled cell growth and division. The disease has the potential to spread to other parts of the body (metastasis) - distinguishing the conditions from benign tumours which stay localised to their area of formation. Whilst traditional treatments include surgery (physically removing the tumour), radiotherapy (killing cells by irreversibly damaging the DNA beyond repair with radiation) and chemotherapy (cytotoxic drugs that kill dividing cells), some drugs currently in clinical trials and development form a category known as targeted therapeutics. Targeted therapeutics identify specific molecular features of the tumour such as specific mutations or base sequences which enables them to exploit the Hallmarks of Cancer. Currently, these drugs are very expensive to develop and deliver - particularly due to the drugs generally only fitting very specific patient cohorts. However, the prospect of more personalised treatment with fewer severe side effects is very appealing to both healthcare professionals and patients.

Some targeted therapeutics are being developed to target the tumour microenvironment. So, what actually is the tumour microenvironment (TME)?

The tumour microenvironment is a dynamic area surrounding a tumour which contains many components including cells and vessels. When inflammation occurs due to oncogenic changes within cancer cells, the internal environment changes. This in turn leads to changes in the surrounding environment - the TME. Tumour cells predominantly display the Warburg phenotype (increased rate of glucose uptake, preferential production of lactate even in the presence of oxygen). However, many tumour cells are still able to use normal metabolic pathways as well as their own. The TME's ability to activate cancer cells even years into the dormancy of the tumour has been noted in many studies. Hence, understanding the TME, and ways in which we can target it, could lead to the development of new targeted therapeutic drugs in the future.

The tumour microenvironment is an immunosuppressive environment, meaning that it debilitates the anti-tumour immune responses through numerous methods, including the following:

• Cancer associated fibroblasts: transfer cysteine, which is converted to glutathione. Glutathione inhibits oxidative stress, which in turn leads to increased resistance against some chemotherapy drugs.
• Angiogenesis: the growth of blood vessels from the existing vessel network of the tumour. This is promoted by an increased concentration of lactate in the TME, which also has an effect on the signalling of cancer-associated endothelial cells.
• Restriction of glucose to T cells (responsible for cell-mediated immunity): an increased concentration of lactate results in the disruption of proliferation of T cells, which in turn affects their function and results in the TME becoming more immunosuppressive.
• Depletion of arginine (as a result of overexpression of the enzyme arginase): the breakdown of arginine and hence arginine depletion within the TME leads to unresponsive T cells, thus increasing the immunosuppressive nature of the TME even further.

As a result of the aforementioned, the TME can be shown to be influenced by tumour cells, leading to development of the tumour by promoting the immunosuppressive nature of its surrounding environment - in this sense, tumours could be seen as self-supporting.

Trabectedin: a targeted therapeutic drug isolated from an anti-neoplastic marine species?

Since targeted therapeutics targeting the TME are fairly new, their impact alone is not fully understood. However, studies are already showing that these drugs may be more effective when used in combination with conventional therapy treatments. What does this mean? Potentially low doses of both drugs, whilst still hitting a threshold to prevent growth of the tumours, which could reduce the chances of any severe side effects otherwise experienced.

Trabectedin was originally isolated from Ecteinoscida turbinata, an anti-neoplastic marine species; contemporarily it is prepared by chemical synthesis. Typically it is used to treat advanced cases of soft tissue sarcomas (STS). However, it has also been used to treat ovarian cancer in conjunction with doxorubicin (a traditional chemotherapy drug).

As shown in Figure 1, trabectedin treats cancer by binding to the minor groove of DNA as a result of locating triplets within the DNA sequence that have a guanine (G) base in the middle of them. Trabectedin covalently binds to one of the two DNA strands and holds the other through hydrogen bonding and van der Waals forces. This:

• blocks transcription through the stabilisation of the double stranded DNA molecule
• inhibits the binding of transcription factors to the DNA molecule, thus preventing cells from replicating their DNA, and leading to the degradation of tumour cells via the proteasome pathway
• interrupts RNA polymerase II during the elongation phase of transcription

Overall, the cells are most sensitive to trabectedin in the G1 phase of the cell cycle (GAP 1), but the drug arrests cells in the G2 and M phases (GAP 2, and mitosis). How was it shown that the microenvironment is the key for trabectedin's mechanism of action? Tumour cells were shown to be resistant to trabectedin in vitro (in cell cultures in petri dishes / in the lab), but sensitive in vivo in the mouse trials conducted - thus showing that only when the tumour microenvironment is present, the drug is effective.

Further developments... do analogues of trabectedin act in a similar way?

Currently, there are ongoing clinical trials involving lurbinectedin - an analogue of trabectedin! This means that the drugs are very similar, both chemically and pharmacologically. There is a very subtle difference in structure between the two which you will be able to identify if you closely observe the top left corner of each drug in Figures 1 and 2.  The mechanisms of action for the drugs are also similar with both targeting both the cancer itself, and the TME. However, lurbinectedin also interacts with mRNA and affects transcription which consequently affects protein production. There are 3 main mechanisms by which lurbinectedin acts as a targeted therapeutic that have been identified so far, as shown in Figure 2:

As shown in Figure 2, lurbinectedin acts via 3 main mechanisms, namely:

1. binding to a CG base pair rich sequence near to, or in, the promoter which leads to the inhibition of transcription as a result of causing the transcription factor to dissociate (transcription inhibition via this method is a common method targeted by chemotherapy drugs already)
2. binding near to RNA polymerase II on the template strand, also leading to transcription inhibition - this form of inhibition is not so widely studied or known about yet
3. lurbinectedin and the protein XPF are both present which prevents nucleotide excision repair, and instead leads to breaks in both single and double stranded DNA molecules, ultimately then leading to apoptosis (programmed cell death)

Other targeted therapeutics you may find interesting!

There are other targeted therapeutics that I have not touched on here which you may find interesting to look at, here are a couple of others:

• herceptin: binds to the HER2 receptor which is overexpressed on abnormal HER2+ breast cancer cells
• iressa (gefitinib): inhibits signalling via Her (EGF) receptors - mutations in EGFR receptor cells renders the cancer cells iressa-sensitive

I hope that through this article I've been able to outline some of the key ideas related to treating cancer using targeted therapeutics - particularly focusing on two drugs, trabectedin and lurbinectedin, the former of which is already being used to treat rare conditions such as soft tissue sarcomas. If you are interested in this area, feel free to ask questions in the "Any Questions?" page and I will reply as soon as possible! In the meantime, look out for another article related to this soon on caspase-8 - the possible link between the tumour and its microenvironment? Stay tuned for more!

References:

• Acikgoz, E., Guven, U., Duzagac, F., Uslu, R., Kara, M., Soner, B. C. and Oktem, G. (2015) Enhanced G2/M Arrest, Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells PLoSOne doi: 10.1371/journal.pone.014109
• Ahmed, N., Escalona, R., Leung, D., Chan., E and Kannourakis., G. (2018) Tumour microenvironment and metabolic plasticity in cancer and cancer stem cells: Perspectives on metabolic and immune regulatory signatures in chemoresistantovarian cancer stem cells Seminars in Cancer Biology doi: 10.1016/j.semcancer.2018.10.002
• Anderson, K.G., Stromnes, I.M. and Greenberg, P.D. (2017) Obstacles posed by the tumormicroenvironment to T cell activity: a case for synergistic therapies Cancer Cell doi: 10.1016/j.ccell.2017.02.008
• Aune, G. J., Takagi, K., Sordet, O., Guirouilh-Barbat, J., Antony, S., Bohr, V. A. and Pommier, Y. (2008) Von Hippel-Lindau-coupled and transcription-coupled nucleotide excision repair-dependent degradation of RNA polymerase II in response to trabectedin Clinical Cancer Research doi: 10.1158/1078-0432.CCR-08-0730
• Balkwill, F.R., Capasso, M. and Hagemann, T. (2012) The tumormicroenvironment at a glance Journal of Cell Science doi: 10.1242/jcs.116392
• Belgiovine, C., Bello, E., Liguori, M., Craparotta, I., Mannarino, L., Paracchini, L., Beltrame, L., Marchini, S., Galmarini, C. M., Mantovani, A., Frapolli, R., Allavena, P. and D’Incalci, M. (2017) Lurbinectedin reduces tumour-associated macrophages and the inflammatory tumour microenvironment in preclinical models British Journal of Cancer doi: 10.1038/bjc.2017.205
• Bensaude, O. (2011) Inhibiting eukaryotic transcription. Which compound to choose? How to evaluate its activity? Transcription doi: 10.4161/trns.2.3.16172
• Bielenberg, D.R. and Zetter, B.R. (2015) The Contribution of Angiogenesis to the Process of Metastasis The Cancer Journal doi: 10.1097/PPO.0000000000000138
• Bueren-Calabuig, J. A., Giraudon, C., Galmarini, C. M., Egly, J. M. and Gago, F. (2011) Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations Nucleic Acids Research doi: 10.1093/nar/gkr512
• Choi, B-S., Martinez-Falero, I. C., Corset, C., Munder, M., Modolell, M., Müller, M. and Kropf, P. (2009) Differential impact of L-arginine deprivation on the activation and effector functions of T cells and macrophages Journal of Leukocyte Biology doi: 10.1189/jlb.0508310
• Cuevas, C. and Francesch, A. (2009) Development of Yondelis(trabectedin, ET-743). A semisynthetic process solves the supply problem Natural Product Reports doi: 10.1039/b808331m
• D’Incalci, M. and Galmarini, C. M. (2010) A Review of Trabectedin (ET-743): A Unique Mechanism of Action Molecular Cancer Therapeutics doi: 10.1158/1535-7163.MCT-10-0263
• D’Incalci, M., Badri, N., Galmarini, C. M. and Allavena, P. (2014) Trabectedin, a drug acting on both cancer cells and the tumour microenvironment British Journal of Cancer doi: 10.1038/bjc.2014.149
• Elez, M.E., Tabernero, J., Geary, D., Macarulla, T., Kang, S.P., Kahatt, C., Pita, A, S-M., Teruel, C.F., Siguero, M., Cullell-Young, M., Szyldergemajn, S. and Ratain, M.J. (2014) First-in-human phase I study of Lurbinectedin (PM01183) in patients with advanced solid tumorsClinical cancer research doi: 10.1158/1078-0432.CCR-13-1880
• Farago, A.F., Drapkin, B.J., Lopez-Vilarinode Ramos, J.A., Galmarini, C.M., Núñez, R., Kahatt, C. and Paz-Are, L. (2019) ATLANTIS: a Phase III study of lurbinectedin/doxorubicin versus topotecan or cyclophosphamide/doxorubicin/vincristine in patients with small-cell lung cancer who have failed one prior platinum-containing line Future Oncology doi: 10.2217/fon-2018-0597
• Forni, C., Minuzzo, M., Virdis, E., Tamboriini, E., Simone, M., Tavecchio, M., Erba, E., Grosso, F., Gronchi, A., Aman, P., Casali, P., D’Incali, M., Pilotti, S. and Mantovani, R. (2009) Trabectedin (ET-743) promotes differentiation in myxoid liposarcoma tumorsMolecular Cancer Therapeutics doi: 10.1158/1535-7163.MCT-08-0848
• Germano, G., Frapolli, R., Belgiovine, C., Anselmo, A., Pesce, S., Liguori, M., Erba, E., Uboldi, S., Zucchetti, M., Paqualini, F., Nebuloni, M., van Rooijen, N., Mortarini, R., Beltrame, L., Marchini, S., Nerini, I. F., Sanfilippo, R., Casali, P. G., Pilotti, S., Galmarini, C. M., Anichini, A., Mantovani, A., D’Incalci, M. and Allavena, P. (2013) Role of macrophage targeting in the antitumor activity of trabectedin Cancer Cell doi: 10.1016/j.ccr.2013.01.008
• Hurley, L. H. and Zewail-Foote, M. (2001) The antitumor agent ecteinascidin 743: Characterization of its covalent DNA adducts and chemical stability Advances in Experimental Medicine and Biology doi: 10.1007/978-1-4615-0667-6_46
• Knowles, H. J. and Harris, A. L. (2007) Macrophages and the hypoxic tumour microenvironment Frontiers in Bioscience doi: 10.2741/2389
• Kouidhi, S., Ayed, F. B. and Elgaaied, A. B. (2018) Targeting Tumor Metabolism: A New Challenge to Improve Immunotherapy Frontiers in Immunology doi: 10.3389/fimmu.2018.00353
• Landskron, G., De la Fuente, M., Thuwajit, P., Thuwajit, C. and Hermoso, M.A. (2014) Chronic Inflammation and Cytokines in the TumorMicroenvironment Journal of Immunology Research doi: 10.1155/2014/149185
• Moneo, V., Martinez, P., de Castro, B., Cascajares, S., Avila, S., Garcia-Fernandez, L.F. and Galmarini, C.M. (2014) Abstract A174: Comparison of the antitumor activity of Trabectedin, Lurbinectedin, Zalypsisand PM00128 in a panel of human cells deficient in transcription/NER repair factors Molecular Cancer Therapeutics doi: 10.1158/1535-7163.TARG-13-A174
• Páez, D., Labonte, M.J., Bohanes, P., Zhang, W., Benhanim, L., Ning, Y., Wakatsuki, T., Loupakis, F. and Lenz, H-J. (2012) Cancer Dormancy: A Model of Early Dissemination and Late Cancer Recurrence Clinical Cancer Research doi: 10.1158/1078-0432.CCR-11-2186
• Patra, J.K., Das, G., Fraceto, L.F., Campos, E.V.R., Rodriguez-Torres, M.dP., Acosta-Torres, L.S., Diaz-Torres, L.A., Grillo, R., Swamy, M.K., Sharma, S., Habtemariam, S. and Shin, H-S. (2018) Nano based drug delivery systems: recent developments and future prospects Journal of Nanobiotechnology doi: 10.1186/s12951-018-0392-8
• Polet, F. and Feron, O. (2013) Endothelial cell metabolism and tumour angiogenesis: glucose and glutamine as essential fuels and lactate as the driving force Journal of Internal Medicine doi: 10.1111/joim.12016
• Santamaría Nuñez, G., Robles, C.M.G., Giraudon, C., Martínez-Leal, J.F., Compe, E., Coin, F., Aviles, P., Galmarini, C.M. and Egly, J-M. (2016) Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells Molecular Cancer Therapeutics doi: 10.1158/1535-7613.MCT-16-0172
• Tumini, E., Herrera-Moyano, E., Martín-Alonso, M.S., Barroso, S., Galmarini, C.M. and Aguilera, A. (2019) The Antitumor Drugs Trabectedin and Lurbinectedin Induce Transcription-Dependent Replication Stress and Genome Instability Molecular Cancer Research doi: 10.1158/1541-7786.MCR-18-0575
• Végran, F., Boidot, R., Michiels, C., Sonveaux, P. and Feron, O. (2011) Lactate Influx through the Endothelial Cell Monocarboxylate Transporter MCT1 Supports an NF-KB/IL-8 Pathway that Drives TumorAngiogenesis Cancer Research doi: 10.1158/0008-5472.CAN-10-2828
• Wermuth, C.G. (2006) Similarity in drugs: reflections on analogue design Drug Discovery Today doi: 10.1016/j.drudis.2006.02.006
• Zhang, W., Trachootham, D., Liu, J., Chen, G., Pelicano, H., Garcia-Prieto, C., Lu, W., Burger, J. A., Croce, C. M., Plunkett, W., Keating, M.J. and Huang, P. (2012) Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia Nature Cell Biology doi: 10.1038/ncb2432