30 January 2023

Fragments in the clinic: MK-8189

Just over seven years ago Practical Fragments highlighted work out of Merck describing the discovery and optimization of potent, selective inhibitors of phosphodiesterase 10A (PDE10A), a potential target for schizophrenia (see here and here). An open-access paper in the latest issue of J. Med. Chem. by Mark Layton and colleagues tells how these were ultimately advanced to a clinical compound.
To recap, a biochemical fragment screen identified the highly ligand-efficient compound 1, which was optimized to the potent compound 2. However, this molecule had poor pharmacokinetics and multiple other liabilities. Further optimization led to Pyp-1, which we noted at the time would make a good chemical probe.

The new paper continues SAR around the central ring, in particular to try to reduce lipophilicity. Also, the methyl-pyrazole in Pyp-1 was associated with high clearance in rats, so this substituent was replaced with a methyl-1,3,4-thiadiazole moiety. To cut a long story short, this ultimately led to MK-8189.
Not only is MK-8189 a picomolar biochemical inhibitor of PDE10A, it is a low nanomolar inhibitor in cells. Moreover, it shows excellent pharmacokinetics in rats and rhesus monkeys as well as selectivity against various off-targets such as CYPs and hERG. Importantly for a drug intended to reach the brain, the molecule is permeable, not effluxed, and achieves relevant concentrations in the rat striatum after oral dosing. Finally, it decreased psychomotor activity and improved episodic memory in rat models of schizophrenia. With all these positives, MK-8189 has been taken into the clinic.
Several lessons emerge from this story. First, as experienced drug hunters will recognize, systematic exploration of multiple positions is important to generate a molecule with the right balance of properties to become an investigational drug. Second, as the researchers note, ligand efficiency was roughly maintained throughout the optimization process. Finally, this publication is a reminder of the long lag that can occur between research and publication. We already mentioned that the first papers describing this series appeared in 2015, but according to ClinicalTrials.gov MK-8189 first entered the clinic a year earlier, in 2014. Our list of fragment-derived clinical compounds will forever be incomplete and out of date. But on a positive note, this means that fragments may be having even more of an impact than the list shows.

23 January 2023

The Chemical Probes Portal at Eight

Back in 2015, Practical Fragments highlighted a new resource calling itself “The Chemical Probes Portal.” At the time it included just seven probes, and my post concluded, “I hope this takes off. Understanding the natural world is hard enough even with well-behaved reagents and carefully controlled experiments.”
Well, take off it has, as illustrated by a new (open access) paper in Nucleic Acids Res. by Susanne Müller (Goethe University Frankfurt), Bissan Al-Lazikani (MD Anderson Cancer Center), Paul Workman (Institute of Cancer Research), and collaborators.
The paper notes that “the widespread use of small molecule compounds that are claimed as chemical probes but are lacking sufficient quality, especially being inadequately selective for the desired target or even broadly promiscuous in behavior, has resulted in many erroneous conclusions in the biomedical literature.” As an antidote, the Portal is an “expert review-based public resource to empower chemical probe assessment, selection, and use.”
Any scientist can suggest a potential probe, and these are then internally reviewed and curated. Assuming enough public information is available about the molecule, probes are then sent to three members of a Scientific Expert Review Panel for further vetting. Reviewers rate probes from one to four stars for use in cellular and/or animal models and recommend relevant concentration ranges. Importantly, reviewers can also include comments to highlight off-targets, lack of certain data, oral bioavailability, or anything else.
From a mere seven probes in 2015 the Portal has grown to include more than 500 molecules covering more than 400 protein targets in about 100 protein families. About two thirds of the probes have three or more stars, meaning they are recommended. The Portal is very easy to use and can be searched by probe or protein. Laudably, all the data can also be easily downloaded in bulk.
In addition to the chemical probes, the Portal also contains around 250 “Historical Compounds” that have been described in the literature but “are not recommended to be used to study the function of specific proteins as they are seriously flawed.” These include molecules such as gossypol, a known aggregator that has been reported as an inhibitor of multiple proteins, and curcumin. If you see a molecule used as a probe in the literature, it’s worth checking to see whether it shows up in the Portal.
The Chemical Probes Portal features heavily in a Conversation between Cheryl Arrowsmith (Structural Genomics Consortium) and Paul Workman published (open access) last year in Nat. Commun. The researchers concisely define chemical probes as “small-molecule modulators to interrogate the functions of their target proteins, as opposed to protein location, or other physical properties.” Importantly, they differentiate chemical probes from drugs. “Drugs don’t necessarily need to be as selective as high-quality chemical probes. They just need to get the job done on the disease and be safe to use. In fact, many drugs act on multiple targets as part of their therapeutic mechanism.” I have frequently heard people make comments such as, “this is just a probe, not a drug,” but a good probe should actually be more selective than many drugs.
That said, you do want a drug to actually hit the target of interest. The researchers highlight iniparib, a putative PARP inhibitor that made it all the way to phase 3 clinical trials for breast cancer and was tested in >2500 cancer patients. It failed. Moreover, that failure cast a pall over the field which likely delayed the development of actual PARP inhibitor drugs.
The researchers also discuss aggregators, which are still being reported uncritically in the literature, along with PAINS. “Such compounds should never be considered further or used as chemical probes. They should be excluded from compound libraries. Yet many are sold by commercial vendors as chemical probes and widely used.”
This statement raised the hackles of Pete Kenny. In a recently published critique, he states: “it is asserted in the conversation that commercial vendors are selling compounds as chemical probes that are unfit for purpose and I strongly recommend that anybody making such assertions should carefully examine the supporting evidence.”
Dear reader, please try the following experiment. Enter “iniparib supplier” in your favorite search engine and see what comes up. For me, the first 10 results include several that describe it as a PARP inhibitor. I won’t link to them here because I don’t want to encourage traffic to their sites. (This is also part of the reason Practical Fragments has discontinued PAINS shaming, as it only increases the profile of sloppy or harmful papers.)
Pete goes on to write: “I would strongly advise against making statements that a compound is unfit for use as a chemical probe unless the assertion is supported by measured data in the public domain for the compound in question.”
Frankly, I don’t understand Pete’s position, which I parodied here. Life is short and biology is complicated, so why waste time with dirty or inadequately characterized reagents? For me, everything is an artifact until proven otherwise. And the Chemical Probes Portal goes a long way towards demonstrating whether a particular probe is fit for purpose.

16 January 2023

Fragments vs SARS-CoV-2: the whole proteome

More than three years have passed since SARS-CoV-2 first entered human airways, and it looks set to stay despite the rapid development of remarkably protective vaccines. Drugs such as nirmatrelvir are effective, but as with any infectious agent we’ll need lots more to counteract inevitable mutational resistance. Practical Fragments has previously discussed virtual and experimental screens against individual SARS-CoV-2 viral proteins and RNA. In Angew. Chem., Harald Schwalbe at Goethe University Frankfurt and more than six dozen collaborators in ten countries describe (open access) the results of NMR screens against most of the viral proteome.
The researchers, all part of the COVID19-NMR project, used ligand-detected NMR methods to screen the DSI-PL library against 25 of the 28 viral proteins. As we’ve written previously, the DSI-PL library consists of 768 diverse fragments designed for rapid chemistry follow-up. Fragments were mostly screened in mixtures of twelve, with spectra visually inspected to identify hits for confirmation.
Fragments were classified as binders if they passed any of these four criteria: “chemical shift perturbations (CSPs) or severe line broadening, sign change in the waterLOGSY (wLOGSY), STD signal or significant decrease of signal intensity in a T2-relaxation experiment.”
A total of 311 hits were identified, with between 2 and 154 hits per protein. In some cases multiple forms of the protein were screened. For example, three forms of the main protease (called by various groups nsp5, Mpro, and CLpro) were screened, yielding from 12 to 78 binders, only 8 of which were common to all three screens. One of the protein constructs forms the biologically relevant dimer (the others are monomers), and the researchers suggest this could account for the differences. True, but I suspect many of the “hits” against some of these proteins are artifacts or non-specific binders: researchers at Vernalis, for example, prioritize fragments that hit in two or three different NMR assays over those that hit in just one.
Crystal structures were available for 18 of the proteins screened, and these were computationally analyzed using FTMap to identify between one and three potential small-molecule binding hot spots on each protein. FTMap uses 16 very small probe molecules (such as benzene and urea) to interrogate the protein surface, and a comparison between the NMR hits with those from FTMap was comfortingly good. For example, a protein with a hot spot preferring benzene and urea also bound a fragment containing those moieties. While this by no means proves that the fragments are binding at a given hot spot, it is suggestive.
Not surprisingly, most of the fragments are weak binders: titration experiments revealed that five of ten tested had dissociation constants > 5 mM, though one came in at double-digit micromolar. This result is consistent with work last year that found that most of the crystallographic hits against Mpro were also weak binders, and also consistent with an independent NMR study of Mpro.
Despite these limitations, this campaign provides multiple starting points to develop chemical probes. Laudably, the chemical structures of all the DSI-PL library compounds and the targets hit by each are provided in the supporting information. Last week we highlighted how fragment hits against the SARS-CoV-2 Nsp3 macrodomain were advanced to sub-micromolar inhibitors. The Angew. Chem. work provides fragment starting points against two dozen more targets.

09 January 2023

From fragments to inhibitors of the SARS-CoV-2 Nsp3 macrodomain

Two years ago we highlighted what was likely the largest crystallographic fragment screen against any target, the macrodomain (Mac1) of the nonstructural protein 3 (Nsp3) of SARS-CoV-2. Mac1 dampens the cellular immune response to viral infection by removing ADP-ribose from various proteins. Separate mutational studies suggested this enzyme could be a good target for treating COVID-19.
The 234 fragment hits identified in 2021 could serve as good starting points. This has proven to be true, as demonstrated in a paper just published (open access) in Proc. Nat. Acad. Sci. USA by Brian Shoichet, James Fraser, and collaborators at University of California San Francisco, University of Oxford, Diamond Light Source, Enamine, and Chemspace. Despite the wealth of fragment hits, none of them were particularly potent; the best had an IC50 value of 180 µM in a homogenous time-resolved fluorescence (HTRF) competition assay. In the new paper, the researchers leveraged computational methods to advance these fragments.
First, they explored a fragment-linking approach termed Fragmentstein. This entailed choosing pairs of fragments that bound in close proximity to one another, merging or linking them, docking them to ensure the new molecule would bind in a similar manner to the component fragments, and then searching make-on-demand libraries in Enamine’s REAL database. Four pairs of fragments were evaluated, and 13 of 16 designed compounds were synthesized. Eight of these confirmed crystallographically, and two showed low micromolar activity in the HTRF assay. Interestingly, both of these came from the same fragment pair, ZINC922 and ZINC337835. The best molecule was a mixture of diastereomers, and one of the pure stereoisomers turned out to be submicromolar.
The potency of this fragment is all the more impressive given the low affinity of the initial fragments, which could only be crystallographically characterized using PanDDA, a method to find low-occupancy ligands that we wrote about here. Unfortunately, the compound has low cell permeability, likely due to the carboxylic acid moiety.
In addition to the linking approach via Fragmentstein, the researchers also conducted two virtual docking campaigns with more than 400 million molecules with molecular weights between 250-350 amu. Of 124 molecules purchased and tested, 47 confirmed crystallographically and 13 confirmed by HTRF, with IC50 values from 42 to 504 µM. (Ten of the 13 HTRF hits were also crystallographic hits. The researchers suggest the difference in confirmation rate is due at least in part to compound concentrations, which were 10-40 higher in the crystallographic screens.) In general the crystal structures confirmed the computationally predicted binding modes, particularly for fragments with measurable activity. Structure-based optimization of some of these fragments led to multiple low micromolar inhibitors, such as LRH-0021. Despite the carboxylic acid, this molecule is cell permeable.

This paper nicely illustrates how even very weak fragments can lead to multiple and very different series of inhibitors. The researchers acknowledge that the molecules are still at an early stage of development; indeed, they note that there are currently no good cellular assays to even assess the effect of Mac1 inhibition. Laudably, all the structures are deposited in the Protein Data Bank, which should provide a useful resource not just for further efforts on this protein but for understanding molecular interactions more generally.

02 January 2023

Fragment events in 2023

Happy New Year!

Several good conferences are scheduled for this year, and while the organizers are hoping for robust in-person attendance there will still be virtual options. Hope to see you at one.

April 11-12: CHI’s Eighteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, is set for sunny San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2022 event here, the 2021 virtual meeting here, the 2020 virtual meeting here, the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting herehere, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here
April 26-28:  While not exclusively fragment-focused, the Ninth NovAliX Conference on Biophysics in Drug Discovery will have several relevant talks. For the first time in five years the event returns to the US (Philadelphia) and and will also offer virtual participation. You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event herehere, and here.

September 25-28: CHI’s Twenty-first Annual Discovery on Target will be held in Boston, as it was last year. As the name implies this event is more target-focused than chemistry-focused, but there are always plenty of FBDD-related talks. You can read my impressions of the 2021 event here, the 2020 virtual event here, the 2019 event here, and the 2018 event here.
Know of anything else? Please leave a comment or drop me a note.