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Applying the "Structured Abstract" principles to evaluating a review article requires a slightly different approach than that used to critically evaluate a primary publication. Let's first establish what makes a good review, an example of which might be this open access review on Cell Cycle modulators. 

Cell-cycle transitions: a common role for stoichiometric inhibitors

Summarise your knowledge of the cell cycle

What is it? Is it universal? How is it regulated? What are the consequences if the cell cycle malfunctions? What are the unanswered questions? 

What is the authors' hypothesis

In this particular review, the authors set out to provide a comprehensive description of the regulatory checkpoints in the cell cycle of eukaryotes. More specifically, they are focusing on information and ideas that surround the activators and inhibitors of the multi-protein networks that define these checkpoints. The authors apply this knowledge to explain the mechanism of "toggling" between states at three checkpoint control sites.

Critical evaluation

These are the kinds of questions you may want to consider. 


  • Does the author(s) provide a few key references that provide you with a link to the landmark discoveries in the field?
  • Is the purpose of the review made clear at the outset?
  • Is there a balanced set of references cited? (You might check how often the review has itself been cited).
  • What is the impact factor for the journal?
  • How productive is the main author (usually the last author in Biology journals)? Search in PubMed (author name and affiliation)
  • Does the review simply list information, or does it offer critical insight?
  • Is there a bias towards the author's own work?
  • Do the figures and narrative (wrt organisation and the quality/accessibility) work for you 
  • Do they try and present an open minded analysis or is it in any way biased


Finally, use the template, but replace the Design/Methods and Results Section with the header 

Organisation and Presentation




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I have been looking over some of your specimen answers, mainly relating to questions that take the form of "describe the mode of action of drug X and the ways in which off target effects are currently monitored". The questions may also be accompanied with the suggestion that "annotated figures" would help. Here are some general suggestions that might help.

1. Before you do anything, you should take some time to read the advice for assessment of examinations given in detail in the MBB handbook. In particular, remember that while recall of factual information forms the foundation of a good mark, it is not sufficient for obtaining a first-class mark. This is discussed in detail in the document.

2. Additional reading is another hallmark of a first-class answer, and with respect to MBB334, my colleagues and I have provided links, attachments and Blog posts, etc to stimulate additional reading. This material is not core to the module, and is not expected to form the basis of the main factual component of an answer to a set question. However, inclusion of extra reading does bring a wider perspective to an answer, and is often characteristic of a more considered and critical answer at level 3, which will often lead to higher marks.

3. Coming to annotated diagrams. Simply put, you can never annotate enough. Please try and keep your diagrams simple (you don't have much time in an exam), but not so simple that they add little or no value to the narrative. At one level an un-annotated diagram of a double helix can speak volumes about the molecule, but labeling the polarity of the strands, the hydrogen bonds, the stacking interactions, the major and minor grooves etc etc, brings a much deeper level of understanding for the reader.

4. Avoid vague statements. Make your writing concise and precise, adding examples to illuminate the narrative, and avoid just saying that you think A is better than B etc. If you do not have a specific point to make, don't waste time on vague generalizations.

5. Finally, as you turn over a page of your answer, to start at the top of the next one, re-read the question, asking yourself if you are "keeping on message"!

I hope this short post offers some help and again, good luck with all or your exams.
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In my last post, I emphasised the problems associated with drug discovery and administration. In this concluding post, supporting my lectures on MBB334, I look at some further examples of drugs that seem to "hit the spot" in a robust and sustainable way, with more caveats, I'm afraid! Hopefully we can learn from our failures as much as we can from our successes. At the foot of this post, there is a short section where I provide some advice on drawing simplified molecular structures in examinations, or simply in a short-hand form. I have also added some comments on the challenges facing the pharmaceutical sector in both drug discovery and safety in an increasingly demanding and rapidly changing global "market place" for health care.

When I am trying to think of drugs that everyone knows, or will have taken, I immediately think penicillin, aspirin, paracetamol, morphine, beta blockers, antihistamines: all of which have been around for at least 40 years (and in some cases longer). Aspirin and paracetamol can both be bought at most supermarkets, while the availability of the others is much more restricted, they are nonetheless familiar to most people. If you search on Google for the highest selling drugs (on a yearly basis) a different picture emerges. Eight out of the top ten drugs (on the basis of cost) are mABs or hormones. If you look at the number of prescriptions per annum, a different picture emerges, reflecting the health care needs/demands of each country. Take a look at the WHO's recommendations for their "essential" medicines: it is published each year, here.

Since the targeted application of monoclonal antibody based drugs has been well documented (see this review for example), I am going to pick a few compounds that are relatively new. The PARP inhibitors target DNA repair and recombination mechanisms and DNA methylation inhibitors (epigenetic drugs), such as decitabine and and zebularine, both target DNA methylating enzymes. These drugs are currently used to treat a number of cancers. The second class of of drugs, the statins, currently "enjoy" a global market value of $20bn, but have recently become "off patent", and therefore the availability of generic versions are set to reduce the market value of statins to the pharmaceutical sector by around 50% (the monthly cost per patient in 2017 was just under £2: the annual cost to the NHS to treat 10% of the population would be approximately £120m!). I shall consider rosuvastatin as an example of a drug that for some can be considered as a "nutritional supplement". Finally I shall say a word or two about the cell cycle inhibitor vincristine as a drug that has been in use for nearly 60 years, prior to a determination of its mode of action. I find the Drugbank website (here is the link to their description of vincristine) to be an excellent springboard for learning more about the mode of action and molecular properties of some (but not all) drugs, and their various commercial formulations. Let's begin with a drug discovery that has a strong Sheffield connection!

Lynparza: its target

PARP, short for poly (ADP-ribose) polymerases, are a class of nuclear enzymes catalysing the modification of target proteins at Glu, Asp and Lys residues, where they attach poly ADP-ribose chains (pADPRs). In addition, PARPs play an indirect, but key role in DNA repair, by binding to single-strand DNA breaks, which in turn leads to the recruitment of repair enzymes. There are just under 20 different PARP proteins expressed from the human genome: PARP1 is the best characterised. It has been shown to play a role several DNA repair pathways including base excision repair (BER) and non-homologous end-joining (NHEJ). The targets for PARP1 include other DNA repair proteins, itself and histones. The structure of PARP1 is shown below: analysis of the sequences of the various PARP family proteins reveals a common set of motifs relating to catalyitic and DNA recognition functions.






How do PARP inhibitors work?


Lynparza (sometimes known as Olaparib) is used to treat cancers of the ovaries, the fallopian tubes and peritoneal cancer. Its chemical structure is shown on the RHS. Once again, it has the usual characteristics of a "drug": aromaticity, several polar groups and (in the case of Lynparza) a cyclopropane moiety at the (right hand) end of the molecule. This compound inhibits PARP activity and as a result, transformed cells, are compromised, leading to a significant reduction in the probability of cancer progression. As discussed in the first part of this post, while it is difficult to design a PARP inhibitor de novo, as with many drug discovery programmes, it is much more likely that subsequent "generation" versions of Lynparza may be more effective. For example, the diagram on the LHS shows a comparison of the binding affinities of 3 PARP inhibitors for a number of PARP variants. A red square indicates high affinity binding (low nanomolar Kd) with progressively weaker binding indicated by orange and yellow. Grey squares indicate the compounds have no specific binding to the PARP indicated. These data highlight some of the limitations of our understanding of the efficacy of drugs of the Lynparza class,  since it may be desirable for an inhibitor to be promiscuous (rucaparib), or highly selective (veliparib) in order to confer a therapeutic benefit, it may also be the case that one of these three contenders exhibits lower toxicity (which can be addressed by cyp450 profiling). I think it is pretty clear that despite a crystal structure for one or two drug-target complexes, a comprehensive understanding of the mode of action of Lynparza will ultimately require a detailed and integrated kinetic and structural analysis of the possible targets in vivo, but in the meantime, Lynparza is bringing considerable benefits to patients, where life expectancy is seriously compromised. 

However.......one of the most frustrating challenges for us all is the phenomenon of drug resistance, and in a recent publication in Nature Communications, a study by Pettitt et al (2018) using CRISPR-Cas 9 technology, have investigated the underlying mechanisms of resistance to PARP inhibitors. It seems that the hopes of new drugs are dashed on the rocks of resistance yet again, but careful stewardship of prescriptions will buy us time until a better molecule is discovered. In summary, while in the case of Lynparza, it is clear that the drug does bring therapeutic benefits, the issues surrounding "resistance" as currently under the microscope with antibiotics) will impact on its future use and will drive the development of new variants.

Zebularine 

Zebularine (and related compounds) were first synthesised around 40 years ago as part of a large programme of medicinal chemistry (at NIH and a number of academic laboratories) centred on nucleotide analogues (see this abstract). Its structure (shown on the RHS) reveals a close similarity with the purine nucleosides (no phosphates) such as cytosine, uracil etc with the notable difference that there is "only" a Hydrogen atom at the C-4 position (12 o'clock). I should immediately mention a very similar analogue to Zebularine called Decitabine. Decitabine is a deoxy derivative (H replaces OH at the 2'C of the sugar ring: at 5 o'clock) of azacytidine: a cytidine analogue in which the ring carbon at the C-5 position (2-o'clock) is replaced by a Nitrogen atom. Both compounds were shown to exhibit "activity" with an early suggestion of a mode of action linked to  enzymes of nucleic acid metabolism. These enzymes initially included cytidine deaminases and thymidilate synthase. However, this has been followed by a more productive focus on epigentic enzymes, including DNA methyltransferases (see this comparative analysis by Judith Christman's group here).  

It seems that Zebularine and Decitabine exert some of their anti-cancer effects by interfering with the normal processes of DNA Methylation. In this respect, azacytidine, has been particularly well characterised by Peter Jones and colleagues, during his many years at the Norris Cancer Center, in Southern California. Those interested may also follow up on the links between chromatin modifications and the possible link to vitamin C, through its role as a modulator of TET: a component of the genome demethylating apparatus in vertebrates. 

[To help in considering drugs that target nucleic acid metabolism, here is a refresher of the terms used with respect to the building blocks of nucleic acids:

Base (eg cytosine, C)
Base + Sugar = nucleoside (Cytidine)
Base + Sugar + Phosphate = nucleotide (eg cytidine monophosphate, CMP), cytidine diphosphate, CDP or cytidine triphosphate, CTP)

[The formulation of drugs like decitabine and zebularine, will be arrived at from an evaluation of their toxic effects, delivery potential, stability etc., which are addressed by the general field of Pharmacology, Toxicology and Pharmacodynamics.]

Both Zebularine and Decitabine are administered in the nucleoside form, anticipating problems with the negative charge from phosphates. Once inside a proliferating cell, the compounds will present as substrates for nucleoside kinases and subsequently RNA and DNA polymerases. This phenomenon in which a drug must be enzymatically modified in vivo, in order for it to exert its effect was called Lethal Synthesis by Sir Rudolph Peters (you can access the information on his original monograph here). I think you will agree, that Decitabine and Zebularine are much easier to reconcile as active site (suicide) inhibitors, compared with Lynparza, for example, when you consider the structural and mechanistic models for the mode of action of DNA methylating enzymes. You can read more about Decitabine and Zebularine at the following links: DNA MTases and Decitabine (original discovery and structural work) and DNA MTases and Zebularine (bacterial and human interactions)

Administration of Decitabine and Zebularine, leads to a low level of substitution of cytosines in both RNA and DNA. In both cases, strong (transiently covalent) interactions occur between the modified base in the target sequence of the enzyme (eg CpG would be ZpG) and the DNA methylating enzymes. This is a consequence of the base flipping mechanism that accompanies targeted DNA methylation. The nucleus is subsequently depleted with respect to DNMTases, as complexed enzymes are degraded, probably via one or more dedicated proteases that are thought to target persistent DNA protein complexes. As a consequence, normal epigenetic programmes in proliferating cells is perturbed, which in turn seems to yield benefits to patients presenting with a number of different tumours.

Vincristine (shown left) is another drug that targets the genome, but unlike Decitabine and Zebularine, its mode of action is indirect: it is mainly deployed in the treatment of non-Hodgkin lymphoma. Vincristine, an alkaloid from the rosy periwinkle,  targets tubulin and thereby preventing normal chromosome segregation, which in turn leads to apoptosis. There are other effects of Vincristine, including an inhibition of leukocyte production. You can read a recent paper here on the current view on the mode of action of microtubule targeting agents, such as vincristine here. I like this poster from the pharmatutor web site.


Drugs versus nutritional supplements?

Stroke patients have sometimes been advised to supplement their diet with a low dosage regime of aspirin. I should point out that there are conflicting views on this advice and the Stroke Association (UK) currently report that the greatest value of aspirin following a stroke is most likely to be when taken in the first few weeks. However, many people swear by taking an aspirin every day. In addition, many people take a single tablet of Rosuvastatin to maintain a healthy cardiovascular physiology. In a sense, these drugs have become nutritional supplements, in the same way that may people of my generation would take the children's vitamin supplement Haliborange from the Seven Seas Company (now a subsidiary of the Pharmaceutical giants Merck). You will be aware of the saying: "an apple a day keeps the doctor away". Apples are generally agreed to be "good for you": they contain a good balance of nutrients, including  fibre, low density sugar and a range of plant secondary metabolites: they also have the value of helping keep your teeth clean (according to my mother). However, they are not formally described as a medicine or a drug. The issue I therefore wish to raise is when does a foodstuff (usually a plant of some sort) become a drug, and when does a drug become a food supplement? I think the answer is simple as Ralph Waldo Emerson once said: Moderation in all things, especially moderation. A nutritionally balanced diet combined with long established drugs and supplements, will, for most people in developed countries, significantly reduce the risk of contracting late life illnesses, such as Type II diabetes. Moreover, this lifestyle strategy, should act as a firm platform for combating and surviving any unexpected infection, serious accident or illness, that will require a more sophisticated drug interventions.

The challenges facing the Pharmaceutical Industry in the future are significant. In the UK alone, we rely heavily on this sector for the drugs we take and for employment (currently over 70 000 jobs, excluding supply chains and related organisations). However, the scientific challenge of "resistance" in its many forms, our limited understanding of both molecular specificity and systems biology, combined with the regulatory hoops through which we demand new drugs to "jump"!, not forgetting the costs and our expectations of eternal life! All of these factors will, in my view, lead to a completely it throughout the second half of the last century. 

Changing tack a little....

Advice on drawing structures in examinations

A number of students have asked me how should they approach the representation of molecular structures under exam conditions. It is an important issue, so I will share some of my own thoughts. Below are two "drugs". One is an antibiotic and the other an anti-hypertensive, statin compound. One compound inhibits the enzyme Chloramphenicol AcetylTransferase (CAT) and the other inhibits HMG CoA reductase. Can you tell which is which? It isn't possible from the (trivial) names: Chloramphenicol and Lovastatin. There is also no easy recognition on the basis of selectivity: a knowledge of the structures of the actual enzyme substrates offers little help (unlike Decitabine and Zebularine). In fact both enzymes have a coenzyme A binding pocket, but we know that subtle changes can completely abolish the inhibitory effect of either molecule. 

These two compounds do share some common features (which is also unhelpful!): both molecules possess an aromatic ring and both have polar hydroxyl and keto groups. In the case of Chloramphenicol there is a conspicuous nitro group, which might be memorable to some of you, but on the whole, most drugs look very similar, but of course not to their respective targets (in the main!). 




Some people can remember the complete structures of many molecules: these individuals are exceptional and if you are such an individual you are lucky. Others will recall the broad features: aromaticity, maybe the competitive binding mode of Lovastatin versus HMGCoA (shown on the RHS). As you can see, there is good steric overlap of these segments of both substrate and inhibitor. But, look up CoA and see how different..
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