Some approaches to design of covalent inhibitors of SARS-CoV-2 main protease

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I last posted on Covid-19 early in 2021 and quite a lot has happened since then. Specifically, a number of vaccines are now available (I received my first dose of AstraZeneca CoviShield in May 2021 while still stranded in Trinidad) and paxlovid has been approved for use as a Covid-19 treatment (Derek describes his experiences taking paxlovid in this post).  The active ingredient of paxlovid is the SARS-CoV-2 main protease inhibitor nirmatrelvir and the ritonavir with which it is dosed serves only to reduce clearance of nirmatrelvir by inhibiting metabolic enzymes. In the current post, I’ll be looking at covalent inhibition of SARS-CoV-2 main protease with a specific focus on reversibility and here are some notes that I whipped up as a contribution to the Covid Moonshot.

Nirmatrelvir (1) is shown in Chart 1 along with SARS-CoV-2 main protease inhibitors from the Covid Moonshot (2), a group of (mainly) Sweden-based academic researchers (3) and Yale University (4).  Nirmatrelvir incorporates a nitrile group that forms a covalent bond with the catalytic cysteine and the other inhibitors bind non-covalently to the target. The first example of a nitrile-based cysteine protease inhibitor that I’m aware of was published over half a century ago and the nitrile warhead has since proved popular with designers of cysteine protease inhibitors (it has a small steric footprint and is not generally associated with metabolic lability or chemical instability). Furthermore, covalent bond formation between the thiol of a catalytic cysteine and the carbon of the nitrile warhead is typically reversible. Here’s a recent review on the nitrile group in covalent inhibitor design and this comparative study of electrophilic warheads may also be of interest.

At this point, we should be thinking about the directions in which design of SARS-CoV-2 main protease inhibitors needs to go. Two directions I see as potentially productive are dose reduction (a course of paxlovid treatment consists of two 150 mg nirmatrelvir tablets and one 100 mg ritonavir tablet taken twice daily for five days) and countering resistance (here’s a relevant article).

Two tactics for achieving a lower therapeutic dose are to increase affinity and reduce clearance. Dose prediction is not as easy as you might think because the predictions are typically very sensitive to input parameters. For example, a two-fold difference in IC50 would often be regarded as within normal assay variation by medicinal chemists but development scientists and clinicians would view doses of 300 mg and 600 mg very differently. 

Excessive clearance is a problem from the perspective of achieving adequate exposure and I'd also anticipate greater variability in exposure between patients when clearance is high. Clearance is clearly an issue for nirmatrelvir because it needs be co-dosed with ritonavir (to inhibit metabolic enzymes) and this has implications for patients taking other medications. Nirmatrelvir lacks aromatic rings and deuteration is an obvious tactic to reduce metabolic lability (although cost of goods is likely to be more of an issue than for a cancer medicine that you'll need to take out a second mortgage for). I would anticipate that bicyclo[1.1.1]pentanyl will be less prone to metabolism than t-butyl (CH bonds tend to be stronger in strained rings and for bridgehead CHs) and the binding mode suggests that this replacement could be accommodated. 

Details of resistance to nirmatrelvir (P2022 | Z2022) are starting to emerge and this information should be certainly be used in design and to assess other structural series. Nevertheless, if you’re genuinely concerned about potential for resistance then you really can’t afford to ignore conserved structural features in the target such as the catalytic residues (cysteine and histidine) and the oxyanion hole. I would also anticipate that the risk of resistance will increase with the spatial extent of the inhibitor.

This post is about covalent inhibitors. Although I’m pleasantly surprised by the potencies achieved for non-covalent SARS-Cov-2 Main Protease inhibitors, I consider making a virtue of non-covalent inhibition to be a serious error. Binding of covalent inhibitors to their targets can be reversible  or irreversible and, in the context of design, reversible covalent inhibitors have a lot more in common with non-covalent inhibitors than with irreversible covalent inhibitors (for example, you can't generally use mass spectroscopy to screen covalent fragments that bind reversibly). In the context of drug design, covalent bonds have much more stringent geometric requirements than non-covalent interactions such as hydrogen bonds.   

I generally favor reversible binding when targeting catalytic cysteines as discussed in these notes and this article. It is typically less difficult to design reversible covalent inhibitors to target a catalytic cysteine than it is to design irreversible covalent inhibitors because you can use crystal structures of protein-ligand complexes just as you would for non-covalent inhibitors. In contrast, the crystal of a protein-ligand complex (the reaction ‘product’) is not especially relevant in design of irreversible inhibitors because target engagement is under kinetic rather than thermodynamic control and the more relevant transition state models must therefore be generated computationally. Furthermore, assays for irreversible inhibitors are more complex, and assessment of functional selectivity and safety is more difficult than for reversible inhibitors. All that said, however, I’m certainly not of the view that irreversible inhibitors are inherently inferior to reversible inhibitors for targeting catalytic cysteines. This is also a good point to mention an article which shows how isosteric replacement (with an alkyne) of the nitrile warhead of the reversible cathepsin K inhibitor odanacatib results in an irreversible inhibitor (the article is particularly relevant if you’re interested in chemical probes for cysteine proteases).

I contributed some designs for reversible covalent inhibitors to the Covid Moonshot and it may be helpful to discuss some of them. Each design was intended to link the nitrile warhead to the ‘3-aminopyridine-like’ scaffold used in the Covid Moonshot which means that the designs all use a heteroaromatic P1 group (typically isoquinoline linked at C4) rather than the chiral P1 group (pyrrolidinone linked at C3) used for nirmatrelvir and a number of other SARS-CoV-2 main protease inhibitors. The ‘3-aminopyridine-like’ scaffold lacks essential hydrogen bond donors (elimination of hydrogen bond donors is suggested as a tactic for increasing aqueous solubility in this article). One of the cool things about the way the Covid Moonshot was set up is that I can link designs as they were originally submitted (often with a detailed rationale and proposed binding mode).

The most direct way to link a nitrile to the ‘3-aminopyridine-like’ scaffold is with methylene (5, Chart 2) but there is a problem with this approach because substituting anilides (and their aza-analogs) on nitrogen with sp3 carbon inverts the cis/trans geometrical preference of the anilides (I discussed the design implications of this in these notes).  This implies that binding of 5 to the target is expected to incur a conformational energy penalty and it is significant that N-methylation of 6 results in a large reduction in potency. Although 5 was inactive in the enzyme inhibition assay, I think that it would still be worth seeing if covalent bond formation can be observed by crystallography for this compound.

However, you won’t invert cis/trans geometrical preference if you substitute an anilide nitrogen with nitrogen rather than sp3 carbon (Chart 3). This was the basis for submitting 8, which is related to azapeptide nitriles, as a design.  Azapeptide nitriles [L2008 | Y2012 | L2019 | B2022] are typically more potent than the corresponding peptide nitriles and, to be honest, this remains something of a mystery to me (one possibility is that the imine nitrogen of the azapeptide nitrile adduct is more basic than that of the corresponding peptide nitrile adduct and is predominantly protonated under assay conditions). I see cyanohydrazines and cyanamides as functional groups that would be worth representing in fragment libraries if you want to target catalytic cysteine residues and I’ll point you toward a relevant crystal structure. The acyclic hydrazine and cyanamide substructures in 8 trigger structural alerts although there are approved drugs that incorporate acyclic hydrazine (atazanavir | bumadizone | gliclazide | goserelin | isocarboxazid | isoniazid) and N-cyano (cimetidine) substructures. The basis for these structural alerts is obscure and it’s worth noting that 8 is incorrectly flagged as an enamine and having a nitrogen-oxygen single bond. As a cautionary tale on structural alerts, I’ll refer you to this comment in which I read the riot act (i.e., the JMC guidelines for authors) to a number of ACS journal EiCs Nevertheless, I’d still worry about the presence of an acyclic hydrazine substructure although these concerns would be eased if each nitrogen atom was bonded to an electron-withdrawing group, as is the case for 8, and all NHs were capped (see 9).

An alternative tactic to counter inversion of the cis/trans geometrical preference is to lock the conformation with a ring and designs 10 and 11 (Chart 4) can be seen as 'hybrids' of 5 with 12 and 13 respectively (in fragment-based design, hybridization is usually referred to as fragment merging). The effect of the conformational lock can be clearly seen since 12 and 13 are essentially equipotent with 6 (the primary reason for proposing 12 and 13 as designs was actually to present the nitrile warhead to the catalytic cysteine). A substituent on carbon next to a lactam nitrogen tends to adopt an axial orientation and I’d anticipate that 10 will be less prone to epimerization than 11. Although I'm unaware of nitriles being deployed on cyclic amine substructures for cysteine protease inhibition, the structures of the DPP-4 inhibitors saxagliptin and vildagliptin are relevant.

This is a good point at which to wrap up. If cysteine protease inhibition is a key component of pandemic preparedness strategy then you really do need to be thinking about covalent inhibition.  I'll be looking at some more design themes for covalent inhibitors of SARS-CoV-2 in the next Covid post.

COVID-19 stuff

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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).