Fragments vs TNFα advanced with biophysics, linking, and growing

The cytokine tumor necrosis factor α (TNFα) is a key mediator of inflammation and has long been a target for rheumatoid arthritis, Crohn’s disease, psoriasis, and a host of other inflammatory diseases. Several biologic drugs, such as adalimumab, are approved but these monoclonal antibodies and fusion proteins can be immunogenic or induce neutralizing antibodies. Small molecules could avoid these pitfalls and also reach organs, such as the brain, less accessible to biologic agents. Impressive efforts towards this goal have just been reported in J. Med. Chem. by Justin Dietrich, Chaohong Sun, and colleagues at AbbVie. (Andrew Petros presented this work at the CHI DoT meeting last September.)

 

The researchers began by screening 18,000 fragments using two-dimensional (¹³C-HSQC) NMR against TNFα in which the methyl groups of isoleucine, valine, leucine, and methionine were isotopically labeled. Only 11 fragments caused significant perturbations, an 0.06% hit rate reflecting the difficulty of finding hits against this target. All the fragments were characterized by SPR, and compound 1 turned out to have reasonable affinity and ligand efficiency. Synthesis of a few dozen analogs led to compound 2, with improved activity.

 

TNFα forms a homotrimer, and a crystal structure of compound 2 bound to TNFα revealed that two copies of the fragment bind within a large hydrophobic cavity at the interface of the three protein monomers. Not present in the apo-form of the protein, this central pocket is formed by the movement of tyrosine side chains, causing desymmetrization of the protein trimer. The researchers linked the two nearby fragments to produce compound 3 with improved affinity but decreased ligand efficiency. Further optimization led to compound 4, which was active in cells. But perhaps not surprisingly given its size and lipophilicity, this molecule had high clearance and poor oral bioavailability in mice. 

 

 

A second fragment, compound 6, had lower affinity than fragment 1, and parallel chemistry efforts generated only flat SAR. A crystal structure revealed that compound 6 bound in a similar manner as compound 1, with two copies in the central cavity. Surprisingly, a crystal structure of compound 8, which differs from compound 6 only by a single methyl group and actually has slightly lower affinity, revealed a singly copy bound in the central void. Scaffold hopping led to compound 9, which was ultimately optimized through structure-based design and careful attention to drug-like properties to compound 12. This molecule is orally bioavailable and showed activity in a mouse arthritis model.

 

This is a lovely paper that illustrates several important lessons. First, as the researchers note, “we have learned from multiple programs, including this one, aimed at developing small-molecule inhibitors of protein-protein interactions, that biophysical methods, when used to drive a fragment-based approach, offer the greatest chance of success.” NMR was essential for finding the initial fragments, SPR provided necessary thermodynamic and kinetic information, and crystallography led to the breakthrough discovery of the binding mode of compound 8.

 

Second, although the initial series generated by fragment linking ultimately did not advance, it proved critical for developing chemical tools, validating assays, and providing structural insights.

 

And finally, this paper is a paean to persistence for difficult targets. As the researchers note, scientists have been seeking small molecule inhibitors of TNFα for decades, and although compounds were reported as early as 2005, most of these have had poor physicochemical properties. Seemingly undruggable targets can sometimes be unlocked. But it usually takes time.

Does configurational entropy explain why fragment linking is so hard?

Linking two weak fragments to get a potent binder is something many of us hope for. Unfortunately, as a poll taken a few years back indicates, it often doesn’t work. But why? This is the question tackled by Lingle Wang and collaborators at Schrödinger and D. E. Shaw in a recent J. Chem. Theory Comput. paper.

 

When a ligand binds to a protein it pays a thermodynamic cost in terms of lost translational and orientational entropy. By linking two fragments, this cost is paid only once instead of twice. In theory this should lead to an improvement of 3.5-4.8 kcal/mol in binding energy, resulting in a 400-3000-fold improvement in affinity over what would be expected from simple additivity. As we noted here, this is possible, though rare. Linker strain often takes the blame as a primary villain. But is there more to the story?

 

The researchers computationally examined published examples of fragment linking (most of which we’ve covered on Practical Fragments) using free energy perturbation (FEP) to try to understand why the linked molecules bound more or less tightly than expected. Impressively, they were able to computationally reproduce experimentally derived numbers, and by building a thermodynamic cycle they could extract the various components of the “connection Gibbs free energy.” These included changes in binding mode or tautomerization, linker strain or linker interactions with the protein, and the previously mentioned entropic benefits of fragment linking.

 

The analysis also identified two additional components. If two fragments favorably interact with each other, covalently linking them may not give as much of a boost. This concept had been considered decades ago, though the current work provides a more general understanding.

 

The more important factor appears to be what the researchers refer to as “configurational entropy.” The notion is that even when a fragment is bound to a protein, both the ligand and protein retain considerable flexibility, which is entropically favorable. Linking two fragments reduces the configurational entropy of each component fragment, and the linked molecule binds less tightly than would be expected. The researchers argue that this previously unrecognized “unfavorable change in the relative configurational entropy of two fragments in the protein pocket upon linkage is the primary reason most fragment linking strategies fail.” They advise that maintaining a bit of flexibility in the linker can help, as has been previously suggested.

 

This is an interesting analysis, and explicitly considering configurational entropy is likely to improve our understanding of molecular interactions. But is it really the main barrier to successful fragment linking? The researchers explore only nine different protein-ligand systems, though they did consider multiple linked molecules for three of these (pantothenate synthetase, RPA, and LDHA). Still, these represent just a fraction of the 45 examples collected in a recent review, and they only considered one somewhat contrived case (avidin) in which especially strong superadditivity was observed. It will be interesting to see whether the analysis holds true for more examples of fragment linking.

Hundreds of fragments hits for the SARS-CoV-2 Nsp3 Macrodomain

COVID-19 will be with us for some time. Despite the unprecedented speed of vaccine development, it is worth remembering that humanity has only truly eradicated two widespread viral diseases, smallpox and rinderpest. Thus, the long march of small molecule drug discovery against SARS-CoV-2 is justified. In a paper recently posted on bioRxiv, Ivan Ahel and more than 50 multinational collaborators take the first steps.

 

Last year we highlighted two independent crystallographic screens against the main protease of SARS-CoV-2. Another potential viral target is the macrodomain (Mac1) portion of non-structural protein 3 (Nsp3), an enzyme which clips ADP-ribose from modified proteins, thus helping the virus evade the immune response.

 

The researchers soaked crystals of Mac1 against a total of 2683 fragments curated from several collections. This yielded 214 hits, and most of the structures were solved at high resolution (better than 1.35 Å). About 80% of the fragments bound in the active site, with many binding in the adenosine sub-pocket. Two different crystal forms were used for soaking, and one set of 320 fragments was soaked against both. Interestingly, this yielded a hit rate of 21% for one crystal form and just 1.3% for the other. Even more surprising, of the five hits found in both crystal forms, only two bound in the same manner in both. This is a clear demonstration that it is worth investing up-front effort to develop a suitable crystal form of a protein before rushing into soaking experiments.

 

Independently, the researchers computationally screened more than 20 million fragments (mostly from ZINC15) against the protein using DOCK3.7, a process which took just under 5 hours on a 500-core computer cluster. Of 60 top hits chosen for crystallographic soaking, 20 yielded structures, all at high resolution (0.94-1.01 Å). The ultra-high resolution structures revealed that four fragments had misassigned structures (wrong isomers), which long-time readers may not find surprising. Importantly, most of the 20 experimentally determined structures confirmed the docking predictions.

 

A strength and weakness of crystallographic screening is that it can find extraordinarily weak binders, which may be difficult to optimize. To see whether they could independently verify binding, the researchers tested 54 of the docking hits in a differential scanning fluorimetry (DSF) assay. Ten increased thermal stability, and all of these had yielded crystal structures. Only four of 19 fragments tested yielded reliable data in isothermal titration calorimetry (ITC) assays, but encouragingly these four also gave among the most significant thermal shifts in the DSF assay. Finally, 57 of the docking hits and 18 of the crystallographic hits were tested in a homogenous time-resolved fluorescence (HTRF) based peptide-displacement assay, yielding 8 and 3 hits respectively, the best with an IC₅₀ of 180 µM.

 

This paper is a tour de force, and may represent the largest collection of high-resolution crystallographic fragment hits against any target. Laudably, all 234 of the crystal structures have been released in the public domain, and the researchers have already suggested ideas for merging and linking. As they point out, many of the fragments bind in the adenine pocket, so selectivity will be an issue not just against human macrodomains but also against kinases and other ATP-dependent enzymes. Still, as the dozens of approved kinases inhibitors demonstrate, achieving selectivity is possible. 

 

From a technology perspective, this publication affirms the rising power of both crystallographic and computational screening. Indeed, the hundreds of crystal structures will themselves be useful input for training new computational methods. And from a drug discovery perspective, each of these fragments represents a potential starting point for SARS-CoV-2 leads.

 

Let’s get busy!

Fragment events in 2021

Gotten vaccinated yet? Don't worry - the first few conferences of the year will be virtual, but hopefully we'll be meeting in person later this year.

March 9-12:  While not exclusively fragment-focused, the Second NovAliX Virtual Conference on Biophysics in Drug Discovery will have several relevant talks. You can read my impressions of the 2018 event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here.

 

May 18-19: CHI’s Sixteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will again be held virtually. This is part of the larger Drug Discovery Chemistry meeting, running May 18-20. You can read impressions of the 2020 virtual meeting here, the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting here, here, 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 27-30: CHI’s Nineteenth Annual Discovery on Target returns to the real world - or at least Boston. 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 2020 virtual event here, the 2019 event here, and the 2018 event here.

December 16-21: What better place to say goodbye to COVID than Hawaii? Postponed from last year, the second Pacifichem Symposium devoted to fragments will be held in Honolulu. Pacifichem conferences are normally held every 5 years and are designed to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, Korea, New Zealand, and the US. Here are my impressions of the 2015 event.

 

Know of anything else? Please leave a comment or drop me a note!