PhD project

A research saga that went through COVID

Research
Drug discovery & design
Author

Jennifer HY Lin

Published

October 23, 2022

The projects

My PhD12 started about 6 months after I’ve finished my MPhil so it was around the beginning of 2015 that I’ve officially begun my PhD and it took a solid 4 years to finish (while working part-time as a hospital pharmacist). In simple words, there were two main research projects going on concurrently in my PhD:

  • SERCA project (details in a separate post as named)
  • Filamenting temperature-sensitive Z protein project (also known as FtsZ project)

FtsZ project was not a new project at the time, but rather a continuation from my previous predecessors in the lab group. Its primary focus was to look for other novel anti-bacterial hit compounds that were different from the ones that were already synthesised and tested in the group. This project was better than the SERCA project in a way that the collaborators were much easier to communicate with even though they were based in India. One down side was that because they’re based overseas, it did take a long time for the compound testing to happen and to have the results returned on time. However, overall, there were much more progress in this project which involved 3 main iterative stages:

Computational work – involved molecular/homology modelling, virtual compound screening (compound size roughly in several 100,000s, please see below links for details) and compound selection for synthesis (using Schrödinger software mainly for this first stage of work)

Compound synthesis – this was certainly the part that has painstakingly taken a lot of time in a chemistry lab out of my four years with many failures in between but with some successes in the end obviously…

Compound testing – it was mainly done by our research collaborators After the completion of these two projects, I was less of a research newbie but I would still humbly call myself a junior researcher, considering how the field of drug discovery can have such an unfathomable depth and breadth…

Image taken by author

The image above was taken literally when I packed up everything after I’ve completed my PhD, which was physically summed into this little box, but what I’ve gained from the journey was far bigger than this. The two hardcover books on the left hand side were my MPhil and PhD theses (potentially useful as treatment for insomnia due to their lengths and time required to read).

Abstract of the projects

Drug discovery is one of the most challenging research fields that contributes to the birth of novel drugs for therapeutic use. Due to the complexity and intricate nature of the research, lengthy processes are involved in identifying potential hit molecules for a therapeutic target. To shorten the time required to reach the hit-to-lead stage, computer-aided drug design (CADD) has been used to expedite the process and reduce laboratory expenses. Common strategies used within CADD involve structure-based drug design (SBDD) and ligand-based drug design (LBDD). Both strategies were used extensively in two projects showing the complementarity of each strategy throughout the process. In this work, two separate drug discovery projects are detailed: Design, synthesis and molecular docking study of novel tetrahydrocurcumin analogues as potential sarcoplasmic-endoplasmic reticulum calcium ATPases (SERCA) inhibitors – details the identification, synthesis and testing of potential hit candidate(s) targeting SERCA by using SBDD Filamenting temperature-sensitive mutant Z (FtsZ) as therapeutic target in ligand-based drug design – details the identification, synthesis and testing of potential hit molecule(s) targeting FtsZ In the first project, homology modelling and virtual compound library screening were utilised as the SBDD methods to identify potential hit molecules for testing in P-type calcium ATPases such as SERCA. Preliminary results have found compound 20, an analogue of tetrahydrocurcumin, to show some SERCA inhibitory effect at 300µM based on a SERCA-specific calcium signalling assay performed via fluorometric imaging plate reader. Molecular docking study has also reflected this outcome with desirable ligand-protein binding energies found for 20 when compared with other tested ligands. Pharmacophore screening was used as the main LBDD method in the second project to identify probable hit candidates targeting FtsZ. Potential ligands were synthesised, and tested for antibacterial effect in Bacillus Subtilis strain 168 (Bs168) and Streptococcus pneumoniae strain R6 (SpnR6) cells. One of the tetrahydrocurcumin analogues, compound 4, was found to have minimum inhibitory concentration (MIC) ≤ 10 µM in Bs168 cells and ≤ 2 µM in spnR6 cells. The IC50 values for 4 were 9.1 ± 0.01 µM and 1 ± 0.01 µM in Bs168 and SpnR6 cells respectively. The MIC of 4 was found to be very similar to the MIC of compound 1, a known hit compound targeting against Bs168 cells. On the other hand, the MIC of 4 was lower than the MIC (> 64 µg/mL) of a well-known FtsZ inhibitor, PC190723, against S. pneumoniae. Subsequent molecular docking analyses were completed to evaluate the ligand-protein binding energies to correlate against the testing results. Both compounds 20 and 4 possess some structural similarities and differences that may confer their different effects in these protein targets, which render both with potentials to become the next lead molecules for future development.

Final update on FtsZ project

As time was ticking along towards the end of 2022, my last attempt to try to get this work published occurred around end of August, where I contacted one of my previous PhD advisers. After that I actually thought there was no way I could carry on in this line of research work, or even think about expanding into cheminformatics for drug discovery and design etc. So in September, I thought about leaving all of this behind and that I need to change direction completely (again), e.g. into health data science or similar. Perhaps it was the universe’s answering or for other unknown reasons, around end of September, an email was sent to me from my previous PhD adviser asking for my help to organise the draft manuscript into the journal template that we were intending to submit. Then in early October, I got another email saying our manuscript was submitted. Then it seemed things started to roll again, after 2019. So this was, indeed, a perfect example showing how a piece of research work was stalled, not only by the commonly known reasons such as delays from collaborating groups, but also by the global-scale pandemic and finally somehow, miraculously, made it to the publication stage.

If anyone asked me if I wanted to go through this again, I would firmly say no as it was mentally painful, but on the other hand, I’ve also gained invaluable things like grit and resilience throughout the process. This has, without doubts, influenced on how I want to approach similar research again, which will be in a different way, in a more data-informed way.

Footnotes

  1. I’ve attempted to communicate with both of my PhD advisers and our research collaborators to publish this work, but to no avail due to the severe restrictions imposed by the COVID-19 situation from the beginning of 2020 and basically also in 2021 (our collaborators were unable to perform any experiments during lockdowns…), a direct publication in a scientific journal is not going to happen any time soon so I thought to provide at least my part to showcase what I’ve done at least…↩︎

  2. Updates on 31/1/2022 – it appeared that my supervisors are in communications with our overseas research collaborators recently about the manuscript so there is a higher chance now that we may be able to publish this work towards the end of 2022 (fingers-crossed…).↩︎