Fragment linking vs IMPDH (with a little help from the literature)

Mycobacterium tuberculosis (Mtb), the cause of its eponymous disease, is making an unwelcome comeback. Current treatments are lengthy, and highly resistant strains are emerging – and spreading. Chris Abell’s lab at the University of Cambridge has been working on multiple tuberculosis targets, and his latest results – in collaboration with Tom Blundell, David Ascher, and collaborators at the University of Cape Town and the University of Melbourne – has just published open access in J. Med. Chem.

The researchers were interested in inosine-5’-monophosphate dehydrogenase, or IMPDH, which is important for the synthesis of guanine nucleotides and is essential for every pathogen examined. They started by screening 960 fragments at 1 mM in a biochemical (spectrophotometric) assay. The IMPDH enzyme from a related organism was used due to its better expression and higher diffracting crystals; selected compounds were cross-checked with the Mtb enzyme and showed similar behavior.

This screen identified 18 molecules that gave at least 50% inhibition. IC₅₀ values were determined for the six most active, and these ranged from 325-675 µM. These molecules were soaked into crystals of IMPDH, but only compound 2 produced a structure. As if to compensate, two molecules of compound 2 bound in the active site. Moreover, these two fragments bound in the same region as a previously reported molecule, compound 1.

Initial attempts at fragment growing yielded only modest improvements in potency, so the researchers tried to link the two copies of compound 2. One linking attempt failed outright, a second gave a 58 µM inhibitor, but a breakthrough came when the linker taken from compound 1 was used. The (S) enantiomer of compound 31 is 2500 times more active than the starting fragment, and crystallography revealed that it binds as designed. Unfortunately, and in contrast to compound 1, compound 31 showed no activity against Mtb in culture. The researchers hope to figure out why.

This paper illustrates several points. First, fragment linking can be quite effective. Second, consistent with our poll from a few years ago, this is not necessarily easy. Indeed, given the reliance on the structure of compound 1, this study can be considered an example of fragment-assisted drug discovery as much as fragment linking. And finally, as we’ve said before, biochemical potency all too often does not translate to cell-based activity – let alone good pharmacokinetic properties. Potency is just the first step in the long march to a drug.

Fragments in the clinic: eFT508

Multiple clinical candidates derived from fragments were described at the recent CHI FBDD Meeting. The story behind one of these has just appeared in J. Med. Chem. in a paper published by Siegfried Reich and colleagues at eFFECTOR Therapeutics.

The researchers were interested in mitogen-activated protein kinase interacting kinases 1 and 2 (MNK1/2), which appear to be important in tumorigenesis but not normal cells. As Paul Sprengeler described it, they started with a “library” of just six fragments – four from the literature and two designed. (The company began in a law office, so hands-on experiments were initially limited.) Some might ask whether this constitutes FBDD, but in the end it’s not the size of your library that counts, but what you do with it.

All the fragments had good affinity, and the researchers were able to obtain crystal structures of four of them bound to MNK2. Optimization proceeded on all six of the fragments, but compound 1 was considered particularly attractive due to its high ligand efficiency and multiple vectors for growing.

Initially the researchers deconstructed the bicyclic ring to compound 7, which led to a 10-fold loss in potency but reduced the molecular weight and lipophilicity. As they note, “loss of potency in exchange for improved physicochemical properties is an often overlooked yet powerful optimization strategy in medicinal chemistry.” Too often people focus on binding over drug-like properties, so it is refreshing to see smart tradeoffs explicitly acknowledged.

Next, the researchers cyclized the molecule to form a lactam and remove one hydrogen bond donor. This also improved the affinity (compound 10). Replacing the phenyl ring in compound 10 with a pyridone in compound 12 further reduced the lipophilicity and improved the selectivity due to non-covalent interactions between a non-conserved cysteine residue and the heterocyclic ring. More optimization led to eFT508.

In addition to low nanomolar potency against both MNK1 and MNK2 in biochemical and cell-based assays, eFT508 is metabolically stable, permeable, and orally bioavailable in mice, rats, dogs and monkeys. The molecule showed good activity in several mouse xenograft models, and tissue samples revealed reduced phosphorylation of the substrate protein eIF4E, as expected. Unlike the MTH1 story last week, in which a selective chemical probe devalidated the target, the results with eFT508 suggest that inhibiting MNK1 and MNK2 has merit, and the compound is currently in four clinical trials for both solid tumors and lymphoma.

Like the story last week, this program progressed rapidly: just 110 compounds, 30 crystal structures, and 1 year to the development candidate. This turned out to be somewhat lucky, as it took another two years to find an equivalently attractive backup candidate. It is also an excellent example of how a non-selective fragment (a purine, found in ATP!) can be turned into a selective molecule. And finally, this is another nice example of how even a public, generic fragment can lead to an attractive chemical series. There are myriad published fragments bound to legions of targets, and it’s worth keeping these in mind whether or not you have in-house biophysical screening capabilities.

Fragments vs MTH1: a chemical probe

As mentioned last week, CHI’s FBDD Meeting was chock-full of success stories. Some of these have recently been published, including work in J. Med. Chem. by Jenny Viklund (Sprint Biosciences) and collaborators at Bayer, the University of Oxford, and the Structural Genomics Consortium.

The researchers were interested in the protein MutT Homologue 1 (MTH1), which helps clear the cell of oxidized nucleotide triphosphates. The enzyme is upregulated in several cancers, and previous research involving non-selective MTH1 inhibitors had implicated it in cancer cell survival. But other research suggested that the effects on cancer cells were due to off-target effects. Clearly what was needed was a high-quality chemical probe.

The researchers started with a thermal shift assay of just 723 fragments screened at 1 mM, of which 166 increased the melting temperature by at least 1°C – a remarkably high hit rate suggesting good ligandability. Of the 49 fragments tested in full dose response thermal shift assays, 48 showed dose dependence. Compound 1 was not the most potent or ligand efficient, but it was synthetically tractable and different from other reported MTH1 inhibitors. Isothermal titration calorimetry revealed a dissociation constant of 49.5 µM, and the compound was also active in an enzymatic assay.

A crystal structure of compound 1 bound to MTH1 guided the selection of similar molecules from an in-house collection, such as compound 3. The structure also revealed a small pocket near the 2-position of the azaindole ring, and compound 5 – also available from the in-house collection – gave a nice pop in potency. Synthesis of a few analogs quickly led to compound 7, with mid-nanomolar activity. Crystallography revealed that the molecule bound mostly as expected. But because an asparagine side chain shifted to accommodate it, standard rigid-protein computational techniques would likely not have predicted its binding.

Further optimization for both potency and DMPK properties ultimately led to BAY-707, which is orally bioavailable in mice. In the interest of space I won’t go into details, but the paper is worth reading for a lovely, well-written account of lead optimization. Astute readers will recognize that all these molecules contain a 7-azaindole core, which is the same moiety that led to three clinical kinase inhibitors. The researchers tested representative molecules against a large panel of kinases as well as other ATPases and determined that the series is quite selective.

With probe in hand, the researchers set off to test whether inhibiting MTH1 would be useful for treating cancer. Unfortunately, as reported in another paper, the results actually “devalidate” the target. Despite potently inhibiting enzymatic activity in cells, BAY-707 showed no growth inhibition on several cancer cell lines, nor did it show activity in mouse xenograft models. While certainly disappointing, the results with this selective inhibitor at least provide a better understanding of biology.

This is also an example of just how quickly FBLD can yield results: at the CHI meeting Jenny said that it took 3.5 FTEs just 14 months from the start of synthesis to discover BAY-707, and the paper says this required only 35 compounds. A nice counterexample the next time someone says fragment approaches take too long.

Thirteenth Annual Fragment-based Drug Discovery Meeting

CHI’s Drug Discovery Chemistry (DDC) meeting was held last week in San Diego. The event continues to grow, and this year hosted some 800 attendees, three quarters from the US and two thirds from biotech or pharma. The first DDC meeting in 2006 had just four tracks, of which FBDD is the only one that remains. The current event had nine tracks and three one-day symposia. There was always something interesting happening, and usually several – at one point three talks involving fragments were going simultaneously. Like last year, I’ll just try to give a few impressions.

What struck me most was the number of success stories, several involving clinical compounds. Last year we highlighted Pfizer’s discovery of a chemical probe against ketohexokinase (KHK); Kim Huard described how this was optimized to PF-06835919, the first and only KHK inhibitor to enter the clinic, which is now in phase 2 trials for NAFLD.

Another phase 2 compound was described by Paul Sprengeler (eFFECTOR Therapeutics). A handful of fragments designed from published work were characterized crystallographically bound to the kinase MNK1, and careful structure-based design resulted in eFT508, an MNK1/2 inhibitor which is being tested against various cancers.

A few years back we highlighted Genentech’s work on the kinase ERK2. In a lovely example of fragment-assisted drug discovery, Huifen Chen told “the convoluted journey of an ERK2 fragment series (with an HTS detour)”. SAR from the fragment series was used to inform the optimization of an HTS series originating from partner Array BioPharma, and was particularly useful for fixing some pharmacokinetic liabilities. Huifen emphasized the importance of using information from multiple strategies, ultimately leading to GDC-0994, which entered phase 1 trials for cancer.

Rounding up the list of clinical compounds, I heard through the grapevine that AbbVie’s dasabuvir, approved for hepatitis C, had fragments in its ancestry. I’d be interested to know more; though since success usually has many fathers, precise parentage can be tricky to ascertain.

Earlier stage success stories included the discovery of BI-9321, a highly selective inhibitor of NSD3-PWWP-1, which binds to methylated lysine residues in proteins. Jark Böttcher described how a collaboration between Boehringer Ingelheim and the Structural Genomics Consortium started with NMR and DSF-based screens of 1899 fragments to identify the cell-active chemical probe.

Jenny Viklund (Sprint Bioscience) described the discovery of potent, selective inhibitors of MTH1, a potential anti-cancer target. The project was successful, but unfortunately the molecules did not have the desired effect in cancer cell lines; this and other evidence helped to devalidate the target. Although undoubtedly disappointing, knowing what not to pursue is still important, and who knows – perhaps the target will turn out to be important in the future.

Finally, Steve Fesik (Vanderbilt) described a number of success stories against the KRAS protein, one of the holy grails of oncology. He also described how a fragment screen against a similarly hot target, the transcription factor MYC, failed utterly – the numerous compounds reported in the literature turned out to be artifacts or DNA intercalators. However, colleague Bill Tansey found that MYC interacts with the protein WDR5, and this protein-protein interaction turned out to be tractable, ultimately yielding potent inhibitors. This is a useful reminder that even if your target is not directly ligandable, biology is complicated enough that you may be able to modulate it through one of its partners.

Success sometimes requires breaking rules, as illustrated by the rule-of-5-defying drug venetoclax. Indeed, as noted by AbbVie’s Phil Cox, 18 of the 76 oral drugs approved since 2014 are bRo5s (beyond rule of 5). But if you’re going to break rules you should expect a harder path, and Phil described factors that correlate with success. Pete Kenny will be delighted to know that this has resulted in a new metric, AB-MPS, which is defined as the sum of the number of rotatable bonds, aromatic rings, and the difference of the ClogD from 3; values less than 12 are correlated with a higher probability of being orally bioavailable among AbbVie’s bRo5s.

Former guest blogger Brian Stockman described NMR-based functional screens he is doing with undergraduates at Adelphi University. Library acquisition can be challenging for a small organization, but happily Dean Brown at AstraZeneca has established an Open Innovation program for neglected diseases – if you’re interested and eligible you can receive a high-quality 1963-fragment library plated and ready for screening.

Of course there was plenty to learn about fragment-finding methods too, both in talks and in a discussion session led by Rod Hubbard (University of York and Vernalis). Microscale thermophoresis (MST) continues to be controversial, with researchers from a couple companies commenting that it’s fantastic the 20% of the time it works, while another company had success rates of ~95%. Thermal shift assays were also contentious, though Fredrik Edfeldt’s (AstraZeneca) method of adding urea or D₂O (see here) to improve the sensitivity created significant buzz.

Cryo-electron microscopy continues to make rapid strides for structurally characterizing difficult targets, such as membrane proteins. Christopher Arthur (Genentech) did not downplay the many technical hurdles, particularly in sample preparation, but he thought that 2 Å resolution structures would be routine within the next decade. Although they have yet to analyze fragment binding, this is only a matter of time.

Ben Cravatt (Scripps) discussed ligand discovery on a proteome-wide scale using electrophilic fragments. His group has currently discovered more than 2000 ligandable cysteine residues in human cells – an exciting if daunting number of potential new targets.

And in the category of now for something completely different, Josh Wand (University of Pennsylvania) described nanoscale encapsulation – in which individual proteins are confined in reverse micelles suspended in liquid pentane; the low viscosity increases tumbling time and thus resolution for NMR, while the miniscule volume increases the concentration of protein and any accompanying fragments. This allows detection of extraordinarily weak interactions (dissociation constants of several hundred millimolar or worse). The technique is limited to very polar fragments because less polar ones would diffuse into pentane, but it would be interesting to see if a fluorocarbon replacement for the hydrocarbon allowed a wider range of fragments to be tested.

I could keep writing but I’ll stop here, hopefully before you stop reading; please leave comments. There are still several good events coming up this year, and mark your calendar for next year, when DDC returns to San Diego April 8-12, 2019!

Universal crystallography

More mature readers may remember a column by Daedalus, aka David E. H. Jones, which used to run in Nature. Sadly he passed away last year, but his company, DREADCO, is still going strong. They have just launched a new product that should be of wide interest.

Our poll last year found that nearly a third of respondents would not begin fragment optimization without a crystal structure. Although there are successful counterexamples, it is fair to say that just about everyone would like a crystal structure if possible. Thus DREADCO has launched UniC, their Universal Crystallography platform.

The idea is based on previous work in which “crystalline sponges” can be used to absorb small molecules. X-ray data are collected on the sponge-molecule complex, and since the sponge structure is already known, the small molecule structure can be readily determined (see here for a nice summary by Derek Lowe). This is a powerful approach for small molecules, but the metal-organic frameworks used for the crystalline sponges are too small for proteins.

DREADCO researchers have solved this problem by using DNA origami to construct a cage-like structure that contains large pores yet is incredibly rigid, and therefore diffracts to high resolution. They have also inserted binding sites for a variety of DNA-binding proteins. All you need to do is generate a fusion between your protein and a DNA-binding protein and soak this into the crystallized DNA cages. Then soak in your fragment, and collect diffraction data to your heart’s content.

UniC is similar to the well-established method of tackling difficult-to-crystallize proteins by generating fusion proteins with antibodies or maltose-binding protein, but there you still need to find and optimize crystallization conditions for the construct. Here, since crystals of the DNA cage can be pre-grown, the time from construct generation to structure determination is dramatically shortened. Whatever the specifics of your protein of interest, all the world’s a cage.

A few years ago Teddy wrote, “The age of the medchemist is over; now is the time of the biophysicist.” Could the same be true for structural biologists who aren’t crystallographers? I hope Teddy puts in a good word for me when he reaches Valinor.