29 March 2020

A crowdsourcing call to action: FBLD vs SARS-CoV-2 Protease

In less than a week the number of cases of COVID-19 worldwide has more than doubled, beyond 720,000, as have the number of deaths, to more than 34,000. For those of us in drug discovery but not on the front lines of clinical care, it is frustrating to watch these numbers climb relentlessly while doing nothing to help other than physical distancing. The temporary closure of so many labs accentuates this feeling.

In early March we highlighted an effort by Dave Stuart, Martin Walsh, Frank von Delft, and others at the Diamond Light Source to screen fragments against crystals of the main protease (MPro) of SARS-CoV-2. The enzyme is a cysteine protease, ideal for covalent fragment screening, and indeed Nir London and coworkers at the Weizmann Institute used intact protein mass-spectrometry to pre-screen 993 fragments. In total, these combined efforts yielded crystal structures of 44 hits bound covalently to the active-site cysteine, 22 non-covalent hits in the active site, and 2 non-covalent hits at the protein dimer interface. Full details and structures can be found here.

In our previous post we showed an overlay of the seven fragments that had been released at the time showing multiple high-quality interactions with the protein. You can look at them all interactively here, and some of the chemical structures are shown below.

This is where crowdsourcing comes in. A group called PostEra (corrected: part of a consortium called COVID MoonShot), consisting of academic and industrial researchers around the world, is trying to use these data and more to develop drugs against SARS-CoV-2. Everyone is invited to contribute, from first year graduate students through industry veterans and emeritus professors.

Do you have ideas how you might grow or merge some of the fragments? If so, you can propose structures, and those that pass a series of filters including synthetic accessibility and toxicity predictions will be synthesized at Enamine and tested at various laboratories (including yours, if you’re interested). We’ve previously highlighted Enamine’s “make on demand” model, which has turnaround times of just a few weeks. At least a couple computational companies, including BioSolveIT and Nanome, are offering free access to their platforms to help you design molecules. Already more than 350 molecule ideas have been submitted.

A cynic could say that these efforts are misguided given the slow pace of drug discovery. Vemurafenib, the first fragment-based drug approved, took six years from the start of the program to approval, and this is lightening speed. However, as Derek Lowe observed, all of the drugs currently being clinically tested against COVID-19 were originally developed for other indications. Stephen Burley suggested recently in Nature that we probably would already have drugs against COVID-19 had we spent more effort fighting SARS.

Hopefully we will have a vaccine long before any drugs coming out of this effort enter the clinic. But there will be a SARS-CoV-3, and a SARS-CoV-4. Having more drugs in our pipeline may prevent those from killing so many people.

23 March 2020

Fragments in the clinic: 2020 edition

As I write this, more than 350,000 people worldwide have tested positive for SARS-CoV-2. More than 15,000 of them have died.

It is important to stay aware of what's going on and take appropriate measures to stop the spread of COVID-19. But to paraphrase Nietzsche, one can spend too much time staring into the abyss. In the spirit of hope, Practical Fragments offers an updated list of FBLD-derived drugs.

The current list contains 47 molecules, 7 more than the last compilation, with 4 approved. As always, this table includes compounds whether or not they are still in development (indeed, some of the companies no longer even exist). Because of this, the Phase 1 list contains a higher proportion of compounds that are no longer progressing. Drugs reported as still active in clinicaltrials.gov, company websites, or other sources are in bold, and those that have been discussed on Practical Fragments are hyperlinked to the most relevant post. The list is almost certainly incomplete, particularly for Phase 1 compounds. If you know of any others (and can mention them) please leave a comment.


PexidartinibPlexxikonCSF1R, KIT
VenetoclaxAbbVie/GenentechSelective BCL-2
Phase 3

Phase 2

AMG 510Amgen KRASG12C
AT9283 AstexAurora, JAK2
IndeglitazarPlexxikonpan-PPAR agonist
MAK683NovartisPRC2 EED
Navitoclax (ABT-263)AbbottBCL-2/BCLxL
Phase 1

ABBV-744AbbottBD2-selective BET
ABT-518AbbottMMP-2 & 9
AT13148AstexAKT, p70S6K, ROCK
AZD5099AstraZenecaBacterial topoisomerase II
BI 691751Boehringer IngelheimLTA4H
HTL0014242Sosei HeptaresmGlu5 NAM
NavoximodNew Link/GenentechIDO1

We live in scary times. But, as this list demonstrates, by working together we can still achieve marvels.

16 March 2020

Fragments vs a Pseudomonas aeruginosa virulence factor

The world is understandably focused on SARS-CoV-2; see for example last week’s post. But there are many other threats out there, including infectious Pseudomonas aeruginosa, which is particularly problematic for immunocompromised people. A recent (open access!) ChemMedChem paper by Martin Empting and collaborators at the Helmholtz Centre for Infection Research and elsewhere describes a clever approach to tackle this pathogen.

An age-old problem for antibiotics is that they provoke resistance: nothing like death to kick evolution into high gear. One way to sidestep this is to develop drugs that target virulence rather than essential microbial pathways. The protein PqsR is part of the Pseudomonas Quinolone Signal Quorum Sensing system, and is important for pathogenicity.

A previously published screen of 720 fragments by SPR yielded about 40 hits, including compound 3. Not only does this compound have impressive ligand efficiency, it also has high enthalpic efficiency; the binding is largely enthalpy-driven. Although the utility of thermodynamics for lead optimization is questionable, the researchers were cognizant of the hydrophobic nature of the ligand binding site for PqsR, and sought molecules that would make polar interactions from the start rather than having to engineer them; a similar strategy proved successful for Astex.

Crystallography with compound 3 was unsuccessful, but SAR by catalog led to compound 7, which has higher affinity for PqsR as assessed by isothermal titration calorimetry (ITC) and also shows activity in a reporter gene assay. Fragment growing led to compound 11, which the researchers were able to characterize crystallographically. The two aromatic rings are at a sharp angle to one another, and attempts at rigidifying the linker proved unsuccessful. But further growing led to compound 20, with submicromolar activity in the reporter assay. This molecule also reduced release of a toxic virulence factor from a clinical isolate of P. aeruginosa.

Interestingly, despite the increased activity of compound 20 over compound 11 in the reporter assay, it seems to have lower affinity for PqsR by ITC. The researchers suggest that the full protein in cells likely behaves differently than the truncated version studied in the biophysical assays.

The researchers also emphasize that flexible linkers were more successful than rigid linkers in improving potency – a phenomenon we’ve previously highlighted here and here. Intuitively a more flexible linker is likely to be more forgiving, as a fraction of an ångström can make the difference between binding or not.

There is still much to do: in particular, activity will need to be improved further, and no pharmacokinetic or other animal data are provided. Moreover, a clinical trial with an anti-virulence strategy would be difficult to design. Still, this is an interesting approach, and I hope the authors or others will follow up on it.

07 March 2020

Fragments vs SARS-CoV-2 Protease: open science in action

Last year we highlighted work done by a consortium called Open Source Antibiotics to find fragment hits against antibacterial targets. A similar effort has now launched to discover leads for COVID-19. And those involved have done so with breathtaking speed and openness.

A group of researchers including Dave Stuart, Martin Walsh, and Frank von Delft (Diamond Light Source) has performed a fragment screen against crystals of the main protease (MPro) of SARS-CoV-2, the virus that causes COVID-19. Even before fully analyzing all of the data, let alone publishing it on bioRxiv, they are making it available here, with promises of frequent updates.

MPro is a cysteine protease essential for viral viability. The first crystal structure of the protein was solved in January and posted on bioRxiv late last month. The Diamond researchers synthesized the gene and used it to produce protein that crystallized and diffracted to high (1.39 Å) resolution. Importantly, they found a crystal form in which the active site was empty and thus well-suited to fragment soaking. In just three days the XChem researchers grew, soaked and analyzed 600 crystals. Since then they have screened over 1000 fragments and found 7 that bind in the active site. These will be released in the protein data bank on March 11, though the coordinates and electron density maps can already be downloaded from XChem and viewed interactively here. An overlay shows a large and attractive pocket with multiple opportunities for protein-ligand interactions.

Frank sent an email on March 6 describing this achievement to a number of researchers, and within minutes Brian Shoichet (UCSF) said that he would be using the fragments as controls in a large library docking screen he is doing. Just a few hours later Andrew Hopkins (Exscientia) said that he has SPR and enzymatic assays up and running and is willing to screen compounds sent to him. Then John Chodera (Memorial Sloan-Kettering Cancer Center) volunteered to do free-energy calculations.

As anyone who has worked in drug discovery, fragment-based or not, will recognize, there is still a long road ahead to turn these fragments into effective drugs. But this global team has sprinted off the starting line. Please join them in the race if you can.

02 March 2020

FBLD meets DEL

FBLD, of course, starts with small libraries of small fragments. DNA-encoded chemical libraries (DEL) usually start from the opposite extreme. Massive numbers of molecules are combinatorially synthesized attached to DNA, screened against a target using affinity selection, and hits identified by sequencing the DNA. A recent paper in J. Med. Chem. by Christopher Wellaway and colleagues at GlaxoSmithKline uses information from both approaches to generate a high-quality candidate.

The researchers were interested in bromodomain and extraterminal (BET) family proteins – the same targets we discussed last week. GlaxoSmithKline had already put molecules into the clinic, but they were looking for structurally different backup candidates, so they performed a DEL screen on the BD1 domain of BRD4. A library of 117 million compounds yielded potent compound 10, and crystallography revealed that the 2,6-dimethylphenol moiety bound in the acetyl-lysine-binding pocket.

Phenols are often metabolic liabilities, and indeed compound 10 was rapidly cleared in mice. However, GlaxoSmithKline has a long and successful history of fragment screening against bromodomains; Teddy first described some of their seminal work back in 2012, when the world didn’t end. Compound 16 had been found in a previous screen as a hit against BRD4, and crystallography revealed that the pyridone binds in a similar fashion to the phenol moiety. (Similar pyridones had been reported by others, for example this one.) Merging the molecules led – after a bit of tweaking – to compound 20a. In addition to BRD4, this molecule binds another bromodomain, BAZ2A, which the researchers wanted to avoid. Structure-based design led them to the more selective compound 20i.

Although compound 20i is potent in cells, it still has moderate clearance in rats. Unsubstituted benzimidazole rings have been reported to be unstable, so the researchers systematically explored a series of substitutions, ultimately arriving at compound 24 (I-BET469). Not only is this compound potent and soluble, it is remarkably stable, with “no detectable turnover in rat, dog, and human microsomal and hepatocyte preparations.” Oral bioavailabilities approach 100%, and the compound proved to be effective in acute and chronic mouse inflammation models. Although selectivity against non-BET family bromodomains members is good, compound 24 does strongly bind to both BD1 and BD2 domains of all four BET family members, and as we saw last week this may lead to toxicity.

Nonetheless, this is a lovely example of using a fragment to replace a problematic moiety in a larger molecule, as we’ve seen previously for chymase, Factor VIIa, and Factor XIa. Throughout the optimization the researchers paid close attention to molecular properties such as lipohilicity and molecular weight, and this resulted in a molecule with excellent pharmacokinetics despite the presence of potentially unstable moieties such as the morpholine. If nothing else, this will be a useful in vivo chemical probe.