Monthly Archives: July 2017

A Day in the Life of a DPhil Student… that also rows for Oxford.

I couldn’t decide whether to write this blog post. However, I sifted through the archives of BLOPIG and found in the original post this excerpt:

“And if your an athlete, like Anna (Dr. Lewis) who crossed the atlantic in a rowing boat or Eleanor who used to row for the blues – what can I say, this is how we roll, or row [feeble attempt at humour] – thats a non-scientific but unique and interesting experience too (Idea #8).  .”

Therefore I’ve decided that it might be an interesting post to look into what life is like when you are studying for a DPhil and also training for the blues. Rowing in particular is a controversial sport – I have heard of many stories advocating that rowing will be the absolute detriment to your DPhil. I’ve never felt pressured as part of OPIG to give up rowing – all of my supervisors have been very fair, in that if I get the work done then they accept this is part of my life. However, I realise all supervisors are not so understanding. I hope this blog post will give some insight into what it is like to trial for a Blues sport (in this case Women’s Lightweight Rowing), whilst studying for a DPhil at Oxford.

4:56 am – Alarm goes off. If its after September it’s dark, cold and likely raining. No breakfast as I will do the first training session fasted – just get dressed and go!

5:15 am – Leave the house with a bag full of kit, food for the day, laptop and papers to cycle to Iffley Sport’s Centre

5:45 am – Lightweight Women’s minibus leaves from Iffley to drive to Wallingford. Some girls try to study in the bus, but to be honest its too dark and we’re all a bit too sleepy.

6:15 am – Arrive at Wallingford. Get onto the water for a session in the boats. Although in the Boat Race we race in an 8 (8 rowers with one oar each, with a cox steering), we spend lots of time in different boats throughout the season. Perhaps unlike our openweight counterparts, we also do a lot of sculling (two oars per rower) as the only Olympic class boat for lightweight women is a sculling boat. We travel to Wallingford for a much longer, emptier stretch of river and normally get to see the sunrise.

 

8:10 am – We leave Wallingford to head back to Oxford. Start waiting in A LOT of traffic once you hit the ring road, and there’s a lot of panic in the bus about whether 9 am lectures will be made on time!

8:50 am – Arrive back at Iffley Sport’s Centre. Grab bike and cycle to the department.

9:00-9:15 am – Arrive at the Department. Quick shower to thaw frozen fingers and to not repulse my fellow OPIG members. I then get to eat warm porridge (highlight of the day) and go through my emails. I also check whether any of my jobs have finished on the group servers – one of the great perks of being in OPIG is the computational resources available to the group. Check the to-do list from yesterday and write a to-do list for today and get to work (coding, plotting results, reading papers or writing)!

11:00 am (Tuesdays & Thursdays) – Coffee morning! Although if it’s any time close to a race no bourbon biscuits or cake for me. This is a bit of an issue because at OPIG we eat a lot of cake. However, one member can usually be relied upon to eat my portion..

1:00 pm – Lunchtime! As a lightweight rower I am required to weigh-in at 59kg on the day of the Boat Race. If I am over that weight I don’t get to race. Therefore, I spend a portion of the year dieting to make sure I hit that target. The dieting lunch consists of soup and Greek yogurt. The post race non-dieting lunch consists of pasta from Taylors, chocolate and a Coke (yum!). OPIG members generally all have lunch at this time and enjoy solving the Times Cryptic Crossword. I’m not the best at crosswords so I normally chat to Laura and don’t concentrate.

2:00 pm – Back to work. Usually coding whilst listening to music. I normally start rushing to be able to submit some jobs to the group servers before I have to leave the office.

3:00 pm – Go to get a chocomilk with Clare. A chocomilk from the vending machine in our department costs 20p and is only 64 calories!

5:30 pm – Cycle to Iffley Sports Centre for the second training session of the day.

5:45 pm – If it’s light enough we hop in the minibus to go to Wallingford for another outing on the water. However, for most of the season its too dark and we head to the gym. This will either consist of weights to build strength, or we will use the indoor rowing machine (erg) to build fitness. The erg is my nemesis, so this is not a session I look forward to. Staring at a screen that constantly tells you how hard you are pushing, or if you are no longer pushing as hard I find to be psychologically quite tough. I’d much rather be gliding along the river.

8:35 pm – Leave Iffley after a long session to head home. Quickly down a Yazoo (strawberry milk) to boost recovery as I won’t be eating dinner until 45 minutes to an hour after the end of the session.

9:00 pm – Arrive home. I “cook” dinner which when I’m dieting consists of chucking sweet potato and healthy sausages from M&S in the oven while I pack my kit bag for the next day.

9:30 pm – Wolf down dinner and drink about a pint of milk, whilst finally catching up with my boyfriend about both our days.

10:00 pm – Bedtime at the latest.

Repeat!

 

When Does Chemical Elaboration Induce a Ligand To Change Its Binding Mode?

When Does Chemical Elaboration Induce a Ligand To Change Its Binding Mode?

For my journal club in June, I chose to present a Journal of Medicinal Chemistry article entitled “When Does Chemical Elaboration Induce a Ligand To Change Its Binding Mode?” by Malhotra and Karanicolas. This article uses a large scale collection of ligand pairs to investigate the circumstances in which elaborations of a ligand change the original binding mode.

One of the primary goals in medicinal chemistry is the optimisation of biological activity by chemical elaboration of a hit compound. This hit-to-lead optimisation often assumes that addition of functional groups to a given hit scaffold will not change the original binding mode.

In order to investigate the circumstances in which this assumption holds true and how often it holds true, they built up a large-scale collection of 297 related ligand pairs solved in complex with the same protein partner. Each pair consisted of a larger and smaller ligand; the larger ligand could have arisen from elaboration of the smaller ligand. They found that for 41 out of the 297 pairs (14%), the binding mode changed upon elaboration of the smaller ligand.

They investigated many physicochemical properties of the ligand, the protein-ligand complex and the protein binding pocket. They summarise the statistical significance and predictive power of the investigated properties with the table shown below.

They found that the property with the lowest p-value was the “rmsd after minimisation of the aligned complex” (RMAC). They developed this metric to probe whether the larger ligand could be accommodated in the protein without changing binding mode. They did so by aligning the shared substructure of the larger ligand onto the smaller ligand’s complex and then carrying out an energy minimisation. By monitoring the RMSD difference of the larger ligand relative to the initial pose (RMAC), they can gauge how compatible the larger ligand is with the protein. Larger RMAC values indicate greater incompatibility, hence a greater likelihood for the binding mode to not be preserved.

The authors generated receiver operating characteristic (ROC) plots to compare the predictive power of the properties considered. ROC curves are made by plotting the true positive rate (TPR) against the false positive rate (FPR). A random classifier would yield the dotted line from the bottom left to the top right, shown in the plots below. The best predictors would give a point in the top left corner of the plot. The properties that do well include RMAC, pocket volume, molecular weight, lipophilicity and potency.

They also combined properties to enhance predictive power and conclude that RMAC and molecular weight together offers good predictivity.Finally, the authors look at the pairs that have low RMAC values (i.e. the elaboration should be compatible with the protein pocket), yet show a change in binding mode. For these cases, a specific substitution may enable formation of a new, stronger interaction or for pseudosymmetric ligands, the alternate pose can mimic many of the interactions of the original pose.

Antibody Developability: Experimental Screening Assays

[This blog post is centered around the paper “Biophysical properties of the clinical-stage antibody landscape” (http://www.pnas.org/content/114/5/944.abstract) by Tushar Jain and coworkers. It is designed as a very basic intro for computational scientists into the world of experimental biophysical assays.]

A major concern in the development of antibody therapies is being able to predict “developability issues” at the screening stage, to avoid costly Phase II/Phase III clinical trial failures. Examples of such issues include an antibody being difficult to manufacture, possessing unsuitable pharmacodynamic or pharmokinetic profiles, having a propensity to aggregate (both in storage and in vivo) and being highly immunogenic.

This post is designed to give a clear and concise summary of the principles behind some of the most common biophysical experimental assays used to assess antibody candidates for future developability issues.

1. Ease of manufacture

HEK Titre (HEKt): This assay tests the expression level of the antibody (the higher the better). The heavy and light chain sequences are subcloned into vectors (such as pcDNA 3.4+, ThermoFisher) and these vectors are subsequently transfected into a suspension of Human embryonic kidney (HEK293) cells. After a set number of days the supernatant is harvested to assess the degree of expression.

2. Stability of 3D structure

Melting temperature using Differential Scanning Fluorimetry (Tm with DSF) Assay: This assay tests the thermal stability of the antibody. The higher the thermal stability, the less likely the protein will spontaneously unfold and become immunogenic. The antibody is mixed with a dye that fluoresces when in contact with hydrophobic regions, such as SPYRO orange. The mixture is then taken through a range of temperatures (eg. 40°C -> 95°C at a rate of 0.5°C/2min). As the protein begins to unfold, buried hydrophobic residues will become exposed and the level of fluorescence will suddenly increase. The value of T when the increase in fluorescence intensity is greatest gives us a Tm value.

(Further reading: http://www.beta-sheet.org/resources/T22-Niesen-fingerprinting_Oxford.pdf)

3. Stickiness assays (Aggregation propensity/Low solubility/High viscosity)

Affinity-capture Self-interaction Nanoparticle Spectroscopy (AC-SINS) Assay: This assay tests how likely an antibody is to interact with itself. It uses gold nanoparticles that are coated with anti-Fc antibodies. When a dilute solution of antibodies is added, they rapidly become immobilised on the gold beads. If these antibodies subsequently attract one another, it leads to shorter interatomic distances and longer absorption wavelengths that can be detected by spectroscopy.

(Further reading: https://www.ncbi.nlm.nih.gov/pubmed/24492294)

Clone Self-interaction by Bio-layer Interferometry (CSI-BLI) Assay: A more high-throughput method that uses a label-free technology to measure self-interaction. Antibodies are loaded onto the biosensor tip and white light is shone down the instrument to yield an internal reflection interference pattern. Then the tip is inserted into a solution of the same antibody, and if self-interaction occurs, then the interference pattern shifts by an amount proportional to the change in thickness of the biological layer. Images from: http://www.fortebio.com/bli-technology.html

(Further Reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896597/)

Hydrophobic Interaction Chromatography (HIC) Assay: Antibodies are mixed into a polar mobile phase and then washed over a hydrophobic column. UV-absorbance or other techniques can then be used to determine the degree of adhesion.

(Further Reading: https://www.ncbi.nlm.nih.gov/pubmed/4094424)

Standup Monolayer Chromatography (SMAC) Assay: Antibodies are injected onto a pre-packed Zenix HPLC column and their retention times are calculated. The longer the retention time, the lower their colloidal stability and the more prone they are to aggregate.

(Further Reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622974/)

Size-exclusion Chromatography (SEC) Assay: Antibodies are flowed through a column consisting of spherical beads with miniscule pores. Non-aggregated antibodies are small enough to get trapped in the pores, whereas aggregated antibodies will flow through the column more rapidly. Percentage aggregation can be worked out from the concentrations of the different fractions.

4. Degree of specificity

Cross-Interaction Chromatography (CIC) Assay: This assay measures an antibody’s retention time as it flows across a column conjugated with polyclonal human serum antibodies. If an antibody takes longer to exit the column, it indicates that its surface is likely to interact with several different in vivo targets.

(Further Reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896597/)

Enzyme-linked Immunosorbent Assay (ELISA) – with common antigens or Baculovirus Particles (BVPs): Common antigens or BVPs are fixed onto a solid surface and then a solution containing the antibody of interest linked to an enzyme (such as horseradish peroxidase, HRP) is washed over them. Incubation lasts for about an hour before any unreacted antibodies are washed off. When the appropriate enzyme substrate is then added, it triggers emission of a visible, fluorescent or luminescent nature, which can be detected. The intensity is proportional to the amount of antibody stuck to the surface.

(Further Reading: https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html)

Poly-Specificity Reagent (PSR) Binding Assay: A more high-throughput method that uses fluorescence-activated cell sorting (FACS), a type of flow cytometry. A PSR is generated by biotinylating soluble membrane proteins (from Chinese hamster ovary (CHO) cells, for example) and then is incubated with IgG-presenting yeast. After washing a secondary labeling mix is added, and flow cytometry is used to determine a median fluorescence intensity – the higher the median intensity, the greater the chance of non-specific binding.

(Further Reading: https://www.ncbi.nlm.nih.gov/pubmed/24046438)