31 July 2023

DNA-encoded fragment growing

When growing fragments into leads, the typical route is making and purifying one compound at a time. In recent years parallel synthesis and screening of crude reaction mixtures has been catching on. However, no physical screening approach can match the throughput of DNA-encoded libraries (DEL). In a new (open access) Chem. Sci. paper, Michael Waring and collaborators at Newcastle University, University of Oxford, and Genentech use DEL to grow fragments.
The researchers started by creating a “poised DNA-encoded library.” We’ve previously discussed the concept of poised libraries, which are designed for rapid follow-up chemistry. Typically, the library members are fragments that contain a handle to facilitate growing. Here, the concept is reversed, with the DEL itself poised to react with a pre-chosen fragment. In this first test case, the DEL consisted of just 42 members made by coupling 7 amino acids to 6 aryl halide-containing acids, which could then be used for Suzuki-Miyaura couplings.
Bromodomains such as the BD1 domain of BRD4 bind a plethora of published fragments, and the researchers chose the 7-atom 3,5-dimethylisoxazole, one of the first fragments published. A boronic acid version of this was coupled to the DEL library and screened against BD1. One DNA sequence in particular occurred 11-times more frequently than any of the 41 others. The corresponding molecule was synthesized without being attached to DNA and found to have a dissociation constant of 51 nM as assessed by NMR. Three control molecules which used different amino acid or aryl-halide building blocks had affinities considerably lower, the best being 2.5 µM.
A crystal structure of compound 22 bound to BD1 showed several important contacts that explain why the molecule was selected in the DEL screen. Moreover, a more lipophilic version of the molecule showed some cell-based activity.
The “NUDEL” (for Newcastle University DEL) is an interesting approach to rapidly explore regions of chemical space around a fragment hit. By including every possible combination of building blocks in the library it is possible to find synergistic combinations, such as those found in compound 22; molecules derived from alanine but a different aryl halide or pyrazole but a different amino acid were not selected over background levels.
Of course, as the researchers acknowledge, 42 compounds is very modest for a DEL. Considerable care was taken to ensure each library member was properly synthesized to facilitate proper analysis (for example, that the selection was not based on differences in concentrations of different library members). This level of care would be more difficult with a million compound library. Also, finding high affinity binders to BRD4 is a rather low bar, particularly when starting with a known fragment. Nonetheless NUDELs look like they could prove quite useful, and I look forward to seeing applications to more novel targets. Perhaps they could even be combined with the DEL-based fragment finding approach we highlighted last year. I predict growing bonds between fragments and DEL.

24 July 2023

Fragments vs VE-PTP: biophysics in action

Protein kinases attach a phosphate group onto amino acid side chains in proteins. Phosphorylation regulates myriad aspects of cell signaling, and thus kinases are common drug targets. Indeed, roughly one third of fragment-derived clinical compounds target kinases. Protein phosphatases remove phosphate groups and thus also make potentially valuable drug targets. Unfortunately, they are very difficult to selectively inhibit, and indeed no fragment-based drugs have entered the clinic. A new paper in Biochemistry from Wataru Asano, Yoshiji Hantani, and colleagues at Japan Tobacco takes the first steps towards rectifying this.
Phosphatases are so difficult to drug because most of them have small, highly charged active sites that have evolved to bind phosphate. This moiety and strongly anionic analogs are not very cell permeable or orally bioavailable. Moreover, the small size of the active site makes selectivity challenging, and the fact that many phosphatases contain an active-site cysteine makes them particularly susceptible to assay artifacts.
The researchers were interested in vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays a role in vascular homeostasis and angiogenesis. They chose 25,000 fragment-sized molecules (with < 20 heavy atoms) from their HTS collection, all with aqueous solubility > 300 µM, and screened these at 250 µM in a mass-spectrometry-based functional assay. Those that inhibited enzyme activity by at least 40% were retested in dose-response format and also characterized by SPR. Many highly acidic compounds such as sulfonic acids were found, but the researchers were particularly intrigued by Cpd-1, which is only modestly acidic with a calculated pKa of 3.9.
Cpd-1 inhibited VE-PTP, but although SPR showed binding, this was not saturable. Thus, the researchers turned to NMR, using multiple protein-observed as well as ligand-observed methods to demonstrate that the molecule binds to the active site of the enzyme. This was confirmed with a crystal structure, which also revealed an “unhappy” water molecule nearby, leading to Cpd-2. This molecule was characterized by crystallography, SPR, and ITC. The molecule proved to be unexpectedly selective for VE-PTP over four other PTPs. The researchers hypothesize that binding to PTPs is often dominated by conserved electrostatic contacts, and because Cpd-2 is less highly charged it relies on other, more specific interactions.
This is a nice example of using a variety of biophysical techniques to find and advance fragments. The researchers do a good job of describing the strengths and weaknesses; for example, it was impossible to determine the dissociation constant of Cpd-1 by SPR due to non-specific binding with the protein, reminiscent of a Pin1 story from several years ago.
There is still a long way to go, with no cell activity or permeability described for Cpd-2. Still, the paper ends boldly: “we believe that this compound will be developed as a potential drug for VE-PTP-related diseases.” Here’s wishing them success.

17 July 2023

A rule of two for using chemical probes?

Earlier this year we highlighted the growth of the Chemical Probes Portal, a free website that profiles more than 500 small molecules targeting more than 400 proteins. Each chemical probe is evaluated by experts based on published literature and then scored for use in cells or in vivo. More than 300 chemical probes have received three or four stars and are thus recommended. But even a good probe can be misused, and this is the subject of a recent (open-access) Nat. Comm. paper from Adam McCluskey, Lenka Munoz, and colleagues at the University of Sydney and the University of Newcastle. (The paper has also been discussed by Paul Workman and Derek Lowe.)
The researchers chose eight probes targeting histone methyltransferases, a histone demethylase, a histone acetyltransferase, and several kinases. All but one of these probes had first been disclosed before 2015. A literature search revealed 662 papers that used these probes in cellular studies, ranging from 21 to 134 publications per probe.
Centuries ago the alchemist Paracelsus noted that everything is poisonous at high enough doses, and indeed even the best probes might hit dozens or hundreds of protein targets. For this reason the Chemical Probes Portal recommends maximum concentrations for cellular assays. The researchers examined whether papers exceeded these concentrations. The overall results were encouraging, with just 22% of papers exceeding recommended limits. However, there was considerable variation: for one chemical probe, 70% of papers exceeded the limit. (For this particular case, the maximum recommended cellular concentration was just 250 nM.)
Because chemical probes can have off-target activity even at recommended concentrations, best practices are to include a related but inactive control compound plus a second chemically differentiated probe. All but one of the eight probes chosen for analysis had orthogonal probes available, and five had inactive controls. So how frequently were these used? Unfortunately, 58% of papers did not use an orthogonal probe, and a whopping 92% of papers did not use available inactive control compounds. In fact, just 4% of the papers “used chemical probes within the recommended concentration range and included inactive compounds as well as orthogonal chemical probes.”
A wider analysis of nearly 15,000 papers that cited the 662 publications produced similar results, with 17% exceeding recommended concentrations, 59% not using differentiated chemical probes, and 83% not using inactive controls.
The researchers propose a “'rule of two': At least two chemical probes (either orthogonal target-engaging probes, and/or a pair of a chemical probe and matched target-inactive compound) to be employed at recommended concentrations in every study.” To encourage best practices, the paper provides a simple “Researchers’ Flowchart” to help investigators select probes and controls. And because science is self-regulated, they provide a five-item “Reviewers’ Checklist.” The paper also includes a nice list of links to other resources, including webinars and slide decks.
Overall I think following these guidelines would be beneficial, and the Reviewers’ Checklist in particular could be usefully incorporated into journal publication requirements.
Of course, the vast majority of protein targets don’t have even a single good chemical probe, let alone two or more. Which means that there are plenty of opportunities to identify new probes and make better use of those that already exist.

01 July 2023

Fragment events in 2023 and 2024

We're at the midpoint of the year but there is still at least one more good conference in 2023, and 2024 is shaping up to be even better.

September 25-28: CHI’s Twenty-first Annual Discovery on Target will be held in beautiful Boston, as usual. 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 2022 meeting here, the 2021 event here, the 2020 virtual event here, the 2019 event here, and the 2018 event here.
March 4-5: RSC-BMCS Ninth Fragment-based Drug Discovery Meeting will be held in Cambridge, UK. This venerable biannual event will be particularly focused on case studies "that have delivered compounds to late stage medicinal chemistry, preclinical, or clinical programmes." You can read my impressions of the 2013 meeting here and the 2009 event here.
April 1-4: CHI’s Nineteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, returns as always (pandemics aside) to San Diego. This is part of the larger Drug Discovery Chemistry meeting. You can read impressions of the 2023 meeting here, 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
September: FBLD 2020 was sadly canceled due to COVID-19, but after a six year hiatus FBLD 2024 is scheduled to be held in Boston (exact dates TBD). This will mark the eighth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2018FBLD 2016FBLD 2014, FBLD 2012FBLD 2010, and FBLD 2009.
Know of anything else? Please leave a comment or drop me a note.