It’s been ages since the last blog post. I’d been thinking of marking my return with an April Fools post but this didn’t seem right given the seriousness of the COVID-19 pandemic. However, I do realize that many people only follow the blog for the April Fools posts so I’ll link them here for easy reference [2013 | 2015 | 2016 | 2017 | 2018 | 2019]. I’m currently in Trinidad so I’ll share a photo from Berwick-on-Sea, on Trinidad's north coast (and the correspondence address for a two [ K2017 | K2019 ] of my more controversial articles).
I should say at the outset that I’ve never previously worked in antiviral area nor tried to help fight a global pandemic. X-ray crystal structures had been published for the main protease of SARS-Cov-2 back in March and these generated some discussion on twitter with Martin Stoermer and Ash Jogalekar (who actually triggered it). The upshot of the discussion was that the discussion was that a hydrogen bond between protein and ligand appeared to be of suboptimal geometry. Martin and I wrote a short article which we uploaded to figshare and Martin also did a blog post. I’ve decided to post my contributions to the COVID-19 response on figshare rather than cluttering ChemRxiv and bioRxiv with preprints that I have no intention of ever submitting to a journal. I should point out the main protease is just one of a number of SARS-CoV-2 targets that one might exploit and I’ll direct you to this helpful review.
The two inhibitors that Martin and I wrote about are both peptidomimetics and each inhibitor structure incorporates a warhead which can form a covalent bond with the catalytic cysteine sulfur. I was particularly interested in the inhibitor with the 𝞪-ketoamide warhead because the inhibition would be expected to be reversible (always a good idea to check though) and I’ll get on to why that’s significant a bit later in the post. When I examine a crystal structure, I first look for what, out of laziness, I’ll call ‘weaknesses’ in the binding mode. These ‘weaknesses’ can be local as is the case for contact between polar and non-polar regions of molecular surface or a hydrogen bond with less than ideal geometry. However, ‘weaknesses’ can also be non-local when a ligand binds in a form (protonation state, tautomer, conformer) that is relatively high in energy. Generally, ‘weaknesses’ in binding modes should always be seen as design opportunities, especially when they are non-local, and here’s an example of how recognition of instability of the bound conformation was used in fragment-based design of PTP1B inhibitors.
It can be helpful to think in terms of design themes when optimizing both hits and leads. Typically, there is insufficient data for building useful predictive models at the start of a project and the optimization process involves efficient generation of the information required for making decisions. As such optimization of both hits and leads should be seen in a Design of Experiments framework. After seeking insights from BB (my mother's dog), I wrote up some design themes.
A crystallographic fragment screen has been run against SARS-CoV-2 and a number of electrophilic fragments were screened using mass spectroscopy. These two screens serve as a launch pad for the COVID Moonshot which looks interesting (although I’d suggest easing off a bit on the propaganda). One limitation of crystallographic fragment screening is that it is very difficult to measure the affinity of fragments which means that it is not generally feasible to explore the structure-activity relationships of fragments prior to structural elaboration. That said, it’s not impossible and I’ll point you to this article which reports a value of -3.1 kcal/mol for the free energy of binding of pyrazole to protein kinase B that was derived from the concentration response of occupancy. The results of the crystallographic screen also have implications for the design of peptidomimetic inhibitors (in particular, the results point to pyridine as a bioisostere for the pyrrolidinone that is commonly used as a P1 substituent) and these some notes may be helpful.
Reversibility is an issue that you definitely need to be aware of when designing compounds to inhibit cysteine proteases and these notes may be helpful. The issue arises because formation of a covalent bond between an electrophilic center (commonly referred to as a ‘warhead’) and the thiol of the catalytic cysteine is a commonly used tactic in inhibitor design. I'll direct you to a review of covalent drugs, an article that discusses some of the things that you need consider when working with covalent inhibitors and a blog post on approved covalent drug mechanisms. There does appear to be a degree of prejudice [R1997 | BH2010 | BW2014] against covalent inhibition and some even appear to be unaware that covalent inhibition can be reversible.
If designing covalent cysteine protease inhibitors, I would generally favor reversible inhibition over irreversible inhibition. My primary reason for taking this view is that design of reversible inhibitors is less complex because IC50 can be interpreted in terms of affinity and you can use pretty much the same structure-based approaches as you would for non-covalent inhibitors. You can't really interpret IC50 for an irreversible inhibitor and the enzyme will be 100% inhibited if it's in contract with an irreversible inhibitor for long enough. The inhibitory activity of irreversible inhibitors is typically quantified by the ratio of the inactivation rate constant (kinact) to the inhibition constant (Ki) which makes the enzyme inhibition assay more complex for irreversible inhibitors. Furthermore, you'll need to build transition state models in order to do structure-based design.
It is possible that irreversible inhibition could lead longer duration of action although you also need to consider the consequences of slow inactivation of the enzyme. If thinking along these lines, you should look at this article by Rutger Folmer. Generally, the decision to go for reversible or irreversible inhibitors is one that drug discovery teams should think through carefully and the decision should determine screening tactics (rather than vice versa).