24 April 2023

RSC Medicinal Chemistry special FBDD issue

The Royal Society of Chemistry puts out RSC Med. Chem., and last year they asked David Rees (Astex), Anna Hirsch (Helmholtz Institute for Pharmaceutical Research Saarland), and me whether a special themed issue on FBDD would be useful for the community. Naturally we said yes, and the results have now been published. You can read our introduction here.
Unlike olden days, when special issues were bound between covers, this is a virtual special issue, with papers published over a period of several months. Indeed, we already wrote about two of them last year: one on combining DNA-encoded libraries (DEL) with FBLD and one on inhibitors of PRMT5/MTA. (Both of these were also topics at the CHI FBDD meeting earlier this month.) In the next few paragraphs we highlight the rest.
AstraZeneca has been doing FBDD since 2002, and has gained hard-won wisdom, some of which was shared in a 2016 review we wrote about here. After years of screening, their fragment library had started to deteriorate, so they rebuilt it entirely, as described by Simon Lucas and colleagues. Some of the starting fragments came from their previous library, but they also considered molecules from their larger collection. Rather than focusing on the rule of three, they developed their own multiparameter optimization function, “FragScore,” which incorporates logD7.4, heavy atom count, number of rotatable bonds, and number of hydrogen bond donors. All compounds were inspected to make sure they would be synthetically tractable, and quality was assessed by SPR, NMR, redox activity, and solubility. The final set consists of 2741 fragments, with a subset of 1152 maximally diverse and attractive fragments for ligandability assessments or screening hard-to-make proteins. They also gathered 16,806 near neighbors for hit follow-up. So far the effort has paid off, with all four of the targets screened thus far yielding progressible hits. If you’re building or renovating a fragment library, you should read this paper.
Continuing on the theme of libraries, Bradley Doak, Martin Scanlon, and colleagues at Monash University describe their “MicroFrag” library, a set of 91 tiny (5-8 non-hydrogen atom) compounds similar to MiniFrags and FragLites. A crystallographic screen (at 1 M concentration!) of the MicroFrag library against the difficult E. coli target DsbA yielded a 52% hit rate, compared with a 2% hit rate with a conventional fragment library. Importantly, the MicroFrag screen identified the two main hot spots previously discovered from the conventional fragment library, along with ten others that may be less actionable. Interestingly, a crystallographic screen of 15 organic solvents at even higher concentrations (50-80%) was less informative: the primary hot spot did not distinguish itself from others. In the case of MicroFrags, not only did this hotspot bind the largest number of fragments, but all the molecular interactions seen for larger fragments were observed.
Fluorine NMR takes advantage of its own specialized library, the subject of a paper by Chojiro Kojima (Osaka University), Midori Takimoto-Kamimura (CBI Research Institute) and collaborators from several institutions. The researchers describe the construction of a 220-member library divided into pools of 10-21 compounds. This library was screened against four diverse proteins, yielding between 3 and 16 hits. The three hits against FKBP were characterized in more detail, including two-dimensional NMR and isothermal titration calorimetry. The researchers also discuss using 19F STD experiments to determine the binding mode of bound fragments.
Fluorine is not the only halogen of interest for library design. We’ve previously described the halogen-enriched fragment library (HEFLib, here and here), which consists of chlorine, bromine, and iodine-containing molecules. Frank Boeckler and collaborators at Eberhard Karls Universität Tübingen and the Max Planck Institute describe screening this library against the Y220C mutant of p53 in an expansion of work they first described back in 2012. Of 14 hits identified by thermal shift or STD NMR, ten confirmed by two-dimensional 1H-15N-HSQC NMR. Four of these bound in the cleft created by the Y220C oncogenic mutation. Two other fragments turned out to be covalent binders, though they reacted with more than one cysteine residue. Although all the fragments have low affinities, they could potentially serve as starting points for optimization.
An ongoing debate is whether there is an advantage to screening more “three dimensional” fragments as opposed to planar aromatic fragments. If your taste tends towards the former, the synthetic chemistry can get tricky. According to an analysis we highlighted last year, the piperidine ring is the third most common scaffold found in drugs. Now, Peter O’Brien (University of York) and an international group of collaborators report efficient synthetic routes to all 20 cis- and trans-piperidines substituted with a methyl group and a methyl ester. A virtual library of 80 compounds in which the secondary amine is capped with simple substituents such as methyl or acetyl groups was found to be quite shapely, particularly compared with the disubstituted pyridyl starting materials. Moreover, the fragments are still reasonably sized, with no more than 15 non-hydrogen atoms and ClogP values < 2.
Machine learning is gaining prominence everywhere, not least in drug discovery. In 2021 we highlighted an “autoencoder” designed for constructing fragment libraries biased towards “privileged” fragments more likely to generate hits. However, the method required considerable programming savvy. Now Angelo Pugliese (BioAscent) and collaborators at the Beatson Institute have implemented their model in the open-source KNIME platform, making it accessible to a wider range of researchers. As an example they use the method to construct a GPCR-focused fragment library, with the structures of all the members provided in the supporting information.
On the subject of fragment libraries, please make sure to vote in our 6-question poll on library design (right side of page; you may need to scroll up).
Not all the papers in this special issue involve library design. Marko Hyvönen, David Spring, and collaborators at University of Cambridge and National University of Singapore describe allosteric inhibitors of the kinase CK2α, which has been implicated in cancer cell survival. We highlighted some of their work against this target in 2017, in which they used fragment linking to find high nanomolar inhibitors of the enzyme. In the new paper, the researchers describe additional fragment binders at the so-called αD pocket, distant from the ATP-binding site. Virtual screening for analogs led to a fragment with mid-micromolar activity in biochemical and cell assays, and fragment merging led to low micromolar inhibitors.
This is a nice collection of papers, and for those of you without easy literature access make sure to check them out soon: for the next six months all of them are free to read after free RSC registration. Enjoy!

17 April 2023

Eighteenth Annual Fragment-Based Drug Discovery Meeting

Last week the CHI Drug Discovery Chemistry (DDC) meeting was held in San Diego, and it was the largest ever, with more than 850 participants, 87% of whom attended in person, up from 70% last year. I won’t attempt to cover all twelve tracks, but will just touch on some of the main themes.
Covalent fragments
Brent Martin kicked off a session devoted to covalent modifiers by describing the in-cell proteome-wide covalent ligand discovery done at Scorpion Therapeutics. Brent emphasized the importance of measuring kinact/Ki to characterize compounds, and he went so far as to say he would recommend rejecting papers that report only IC50 values. He emphasized some of the challenges finding low-affinity fragments (with high Ki values) that are not overly reactive. But Upendra Drahal (Amgen) noted that sotorasib has only weak affinity (Ki = 86 µM) but a high kinact (0.85 s-1) for the G12C mutant form of KRAS, despite being quite stable against glutathione. High reactivity is fine, as long as it is highly selective reactivity.
Jeffrey Martin (Biogen) spoke about covalent fragments applied to neuroscience, including targets for Alzheimer’s (tau) and Huntington’s disease. And in two separate talks Dan Nomura (UC Berkeley) provided numerous examples of finding covalent fragments against a variety of targets, including cMYC and E3 ligases (more on those below).
Keynote speaker Michelle Arkin (UCSF) described using disulfide Tethering to find reversible covalent binders of caspase 6 that were subsequently optimized to cell-active irreversible inhibitors. She provided an evocative visual metaphor of proteins participating in an English country dance, moving from partner to partner in a dynamic yet choreographed fashion. The most popular dancers are the 14-3-3 proteins, which act as hubs mediating binding to hundreds of other proteins. Michelle has found stabilizers of some of these interactions, which could shut down aberrant disease signaling.
Covalent ligand discovery is best done with mass spectrometry, and two talks revealed useful new methods. Jim Nonomiya described an approach he and his Genentech colleagues developed called CoMPAS, covalent mapping by peptide attenuation screening. This method uses an isotopically labeled peptide as an internal standard to assess disappearance of covalently modified peptides from enzymatically digested proteins. Sensitivity can be much better than for intact protein mass spectrometry, allowing lower consumption of scarce recombinant protein. Depending on the type of setup, throughput can also be higher.
Throughput is the name of the game in a method presented by Nate Elsen (AbbVie) called IR-MALDESI-MS. The home-built system uses a laser to gently ionize aqueous solutions in 384-well plates before running them through an electrospray mass spectrometer. The system can analyze up to 20 samples per second, including intact proteins.
Non-covalent fragments
Turning to non-covalent methods, Rod Hubbard (Vernalis) provided an update of the PAC-FragmentDEL approach which we highlighted last year. DNA-encoded libraries can be mind-bogglingly large, with more than a trillion molecules at Hitgen. The fragment set is much smaller, at just 130,000 members, but this is still two orders of magnitude larger than a typical fragment library. (Speaking of which, please make sure to fill out our library survey on the right-hand side of the screen if you haven’t already done so.) This increased chemical diversity increases the odds of finding rare molecules, such as molecular glues that bind to protein complexes rather than individual components.
Success stories are always plentiful at conferences. Timo Heinrich (EMD Serono) described using SPR to identify fragments that were optimized to orally bioavailable inhibitors of the anti-cancer target TEAD1, which we wrote about here. Mihir Mandal (Merck) described the use of fragment concepts in the development of clinical candidates targeting metallo-β-lactamases, an important cause of antibiotic resistance. Chris Smith described Mirati’s discovery of inhibitors against the anti-cancer target PRMT5/MTA, one of which has gone into the clinic (see here and here). And Tanweer Khan traced the origins of renin and plasmepsin inhibitors to work on the aspartyl protease BACE1 at Merck, which we wrote about here.
Sometimes we learn just as much from projects that don’t move forward, as illustrated in a nice talk by Haihong Wu. He and his AbbVie colleagues were interested in the tau protein, which is intrinsically disordered, making structure-based design difficult. Although a two-dimensional NMR screen identified several dozen fragment hits, these were hard to optimize, with sharp, non-linear SAR. Perhaps the covalent binders we mentioned above will be more advanceable.
Targeted protein degradation
Targeted protein degradation and induced proximity were major themes of the conference. We speculated several years ago that FBLD could be useful here, and this has turned out to be abundantly true.
E3 ligases attach ubiquitin to other proteins, marking them for degradation. More than 600 exist in the human genome, but only a handful have been co-opted for targeted protein degradation, and everyone is racing to find new and better ligands for the unexplored E3 ligases. Mary Matyskiela described how she and her colleagues at Neomorph are using fragment screening to identify such ligands. She said they have been able to use cryo-electron microscopy to guide structure-based design.
Dan Nomura’s second talk focused on finding ligands against E3 ligases, including UBE2D and RNF126. For both of these proteins small covalent ligands seem to be generally useful for causing degradation of target proteins, and because the chemical structures have been disclosed it will be fun to see them explored in more systems.
A significant focus for targeted protein degradation is to find ligands for E3s that are restricted to specific tissues or tumors. Steve Fesik (Vanderbilt) described using SAR by NMR to find ligands against three E3 ligases, including one not expressed in the heart, which could avoid cardiotoxicity. Steve also described using SAR by NMR to find nanomolar binders for β-catenin. These are being used to make degraders for this anti-cancer target.
Many of the molecules used for targeted protein degradation are well beyond conventional rule-of-five space as they contain binding moieties for the target of interest as well as an E3 ligase. Reflecting on his forty-year career as a medicinal chemist at Bristol Myers Squibb, keynote speaker Nicholas Meanwell noted that a beautiful drug is one that helps patients, not one that fits a set of metrics. For small molecule therapeutics, he observed, “the opportunities have never looked better.”
We’ll end on this note, but please feel free to leave comments about your highlights. And mark your calendar for April 1-4, when DDC returns to San Diego.

10 April 2023

New poll: fragment libraries in 2023

The quality of a fragment hit depends on the quality of the library. Virtual screens can be done with billions of compounds, but if you’re going to build a physical library you need to be pickier. Researchers today are increasingly avoiding pathological molecules such as PAINs, but even sticking to “reasonable” molecules leads to choice overload.
So for those building or refurbishing their libraries, Practical Fragments launches a new poll. It’s been almost five years since we’ve asked readers about their libraries, and the current poll is our longest yet, with six questions.
The first three questions focus on size: how large is your fragment library and what are the minimum and maximum number of heavy atoms in each fragment.
Next, we ask about whether you include chiral molecules in your library, and whether these are present as enantiomerically pure compounds or racemates.
Chiral compounds can introduce synthetic challenges which may impede hit follow-up, so our next question asks whether you consider synthetic tractability before adding fragments to your library.
Finally, building a proper fragment library requires considerable time and resources, so you want to take good care of it. Our last question probes how you store your fragment library. Note that this question asks about your working library, the one that you screen and access on a regular basis, not the master stocks which may be squirreled away as solids in the deep freeze.
Please vote on the right-hand side of the page. If you have multiple fragment libraries (perhaps one for crystallographic screening and another for biochemical screening) feel free to vote for each; you need to press "Finish Survey" at the end.
There are, of course, more complex elements to library design that can’t be captured in simple multiple-choice polls. For example, we’ve written previously about the importance of function rather than functional groups, and the types of rings found in approved drugs. If you have opinions about these or other subjects, please share.
The poll was already long so we decided not to ask about library vendors, which we addressed in 2018. On this topic too, please feel free to share your thoughts.
Happy voting!

01 April 2023

Profitable Fragment$

Practical Fragments has always been free and worth every penny. But over the past 15 years and 900+ posts we believe we've generated a lot of value, so today we're launching our for-profit sister site, Profitable Fragment$. Here you'll be able to buy all your favorite branded items, such as Sauron Atoms jerseys, Voldemort Rule mugs, and Dr. Saysno baseball caps. But the real moneymaker, we believe, is our new line of NFTs.
Non-fungible tokens, or NFTs, are one-of-a-kind digital thingamajigs that represent ownership of something else, such as works of art. And what are molecules if not works of art? Every drug that has ever been invented will have an NFT. But Profitable Fragment$ has no intention of stopping there. No, we intend to create NFTs for every fragment ever published. Fragments that have led to drugs, or even those with associated crystal structures, will go for a premium.
We'll also have a special line of PAINS NFTs, akin to Garbage Pail Kids. Will toxoflavin sell for more than vemurafenib?
Even if you can't afford something fancy like sotorasib or 7-azaindole, don't despair: there are nearly 170 billion cheaper options drawn from GDB-17. Heck, if these sell for just 10 cents per fragment it should generate more than enough cash for us to buy back Twitter!