Bioisosterism surprises

The concept of bioisosterism is central to medicinal chemistry. Essentially, one functional group is replaced by another which has similar activity but a different chemical structure. This might be done for a variety of reasons: improving pharmaceutical properties, enabling new analogs, or inventing around existing intellectual property. Most medicinal chemists are familiar with common bioisosteres, such as replacing a carboxylic acid with an acyl sulfonamide. But what about replacing a carboxylic acid with an amidine? This and other surprising examples are provided in a new Angew. Chem. paper by Gerhard Klebe and colleagues at Philipps Universität Marburg.

 

The researchers focused on fragments binding to the hinge region of protein kinase A (PKA), a well-characterized and easily crystallized kinase. As we noted a couple weeks ago, most kinase inhibitors bind to the so-called hinge region, where the adenine ring of ATP normally sits. Protein backbone amides typically make one to three hydrogen bonds with inhibitors. The researchers chose 19 simple fragments, each containing an aromatic ring and various substituents, soaked these into crystals of PKA, and obtained high-resolution (between 1.12 and 1.82 Å) structures. They also experimentally measured the pKa values of each fragment.

 

All except two of the fragments made one or two hydrogen bonds to a backbone amide NH and/or carbonyl oxygen, but the moieties that did so varied dramatically. Benzamide, with its hydrogen bond accepting carbonyl oxygen and hydrogen bond donating primary amide, is a quintessential hinge-binder, but surprisingly benzoic acid bound in a similar fashion. The measured pKa of this carboxylic acid is 4.01, yet the acid serves as a hydrogen bond donor, suggesting that it is protonated in the active site of the enzyme.

 

On the other end of the acidity spectrum, a substituted benzamidine fragment with a pKa of 10.78 bound in the neutral form, with a normally charged nitrogen atom serving as a hydrogen bond acceptor. In fact, the binding mode it assumes is identical to that of benzoic acid.

 

These and several other examples illustrate that protonation states of ligands in active sites can be very different from what one would predict based on calculated or even measured pKa values. There are of course limits: an amidine with a measured pKa of 11.32 avoids the hinge and instead interacts with an aspartic acid side chain.

 

One quibble is that the researchers did not seem to consider hydrogens on carbon atoms as potential acceptors; these are increasingly recognized as important, including in kinases. One pyridine fragment shown may have a CH in close proximity to a carbonyl, but it is difficult to tell from the figures, and the coordinates have not yet been released.

 

Another omission is the lack of quantitative information about binding energies. Just because benzoic acid and a benzamidine bind identically does not mean they have the same affinities. That said, Gerhard Klebe warned last year of the dangers of putting too much stock in thermodynamic measurements.

 

These issues aside, this is a nice analysis and should serve as a useful reminder to medicinal chemists that bioisoteres can be quite unexpected. And once the structures are released in the pdb, they will provide a useful resource for modelers seeking to recapitulate crystallographic data.

Massive crystallographic drug screen against SARS-CoV-2 main protease

As of November 23, more than 58 million people worldwide have contracted COVID-19, and more than 1.3 million have died. Each of these numbers is roughly two orders of magnitude higher than in this post published exactly eight months ago. Progress towards vaccines and biological treatments has been stunningly fast, but small molecules could still play a role. Towards this end, Sebastian Günther, Alke Meents, and nearly 100 collaborators from the Center for Free-Electron Laser Science at DESY and multiple other institutions have just posted a preprint on bioRxiv.

 

The researchers were interested in drug repurposing, in which approved or clinical-stage molecules are tested against a new target. Typically this is done in some sort of biochemical or cell-based assay, but in this case the researchers chose crystallographic screening against the main protease (M^pro) from SARS-CoV-2. An independent fragment screen against the same target was published recently in Nat. Comm. (I wrote a companion Comment, and both articles are open-access.)

 

The current screen of 5953 compounds may be the largest crystallographic screen in history, and the first I know of that used drug-sized molecules rather than fragments. Even more impressive, all compounds were co-crystallized with M^pro, a much more tedious process than the usual soaking. The advantage of co-crystallization is that the protein is more able to change conformation in response to compound binding, but the disadvantage is that small molecules may prevent crystallization. Ultimately 3955 compounds allowed crystal formation, of which 3228 produced crystals that diffracted better than 2.5 Å, and 1196 produced usable datasets. The result? Just 37 unique binders, or 0.6%. Comparing this to the 96 fragment hits from the smaller fragment library is complicated by differences in methodologies, but it does seem likely that molecular complexity played a role in the lower hit rate: the median molecular weight of the drugs screened, 366.5 Da, comfortably exceeds fragment space.

 

Among the 37 binders, only 29 gave sufficiently well-resolved electron density to determine binding modes. Of these, ten bound covalently. The catalytic cysteine seems particularly reactive, as evidenced by the fact that seven structures showed maleate – a common pharmaceutical counterion – covalently bound. One of the covalent molecules, calpeptin, is a cysteine protease inhibitor that had previously been reported to be active against SARS-CoV-2, but the others are less predictable. In addition to the active site, some molecules bound to two possibly allosteric sites.

 

Crystallographic hits were tested for inhibition of viral replication in cells. Ten were active, and a few (calpeptin, pelitinib, and isofloxythepin) had single digit micromolar activity. Interestingly, despite being designed as a covalent kinase inhibitor, pelitinib binds noncovalently. In contrast, isofloxythepin, a non-covalent dopamine receptor antagonist, binds covalently.

 

In addition to the cell-based screen, many of the compounds also showed binding by native electrospray ionization mass spectrometry (ESI-MS). However, as we’ve noted previously, the correlation between affinity and ESI-MS binding can be tenuous. It would be nice to see the affinity or activity of the compounds via a more quantitative method. Indeed, the researchers note that none of the non-peptidic molecules had previously been reported as M^pro inhibitors, so they may be quite weak. Another problem is that – in contrast to the crystallographic fragment screen – none of the coordinates seem to have been released yet. Hopefully this will be rectified when the paper is formally published.

 

This campaign is yet more evidence that crystallography has come into its own as a primary screening methodology. The researchers note that they “now routinely measure 450 datasets per day,” with a goal of reaching 1000. Whether or not these results impact the course of COVID-19, the techniques developed will likely impact future drug discovery efforts.

Kinase fragments galore: a free virtual collection

Kinases hold a special place in fragment-based drug discovery. Vemurafenib, the first approved FBDD-derived drug, targets a kinase, as do more than a third of fragment-derived drugs to enter the clinic. These efforts have produced a wealth of knowledge, and in a new paper in J. Chem Inf. Mod. Andrea Volkamer and collaborators at Universitätsmedizin Berlin and Bayer have extracted thousands of virtual fragments and made them freely available in a database called KinFragLib.

 

The researchers started with a prior database called KLIFS, which compiles thousands of crystal structures of kinases bound to small molecules. Kinase inhibitor binding modes are classified into several  types, and to keep things simple the focus here was on Type I and Type I¹/₂. Both bind to the active, DFG-in form of the kinase, the only difference being that Type I¹/₂ binders extend into a back pocket. A total of 2801 crystal structures were selected for analysis.

 

Next, the ligands were computationally fragmented using a methodology called BRICS (Breaking of Retrosynthetically Interesting Chemical Substructures). Molecules such as ATP and other substrate analogs were discarded to keep the focus on drug-like compounds, and some particularly large, complex molecules such as staurosporine could not be fragmented. The researchers were particularly interested in molecules that bind to the so-called hinge region, where the adenine moiety of ATP binds, so the few ligands that did not bind here were also removed. This reduced the total number of structures to 2553, which yielded 7486 fragments.

 

The kinase active site was divided into six sub-sites: the adenine pocket, solvent-exposed pocket, front pocket, gate area, and two back pockets. Each of the fragments was then assigned to one sub-pocket. More than 80% of the original (unfragmented) ligands bound to two or three sub-pockets, while another 13% bound to four sub-pockets. Just 5% of the original ligands bound only to the adenine sub-site, but these 127 ligands – with an average of 15 non-hydrogen atoms – could be quite interesting as crystallographically validated fragments.

 

Various analyses of the fragments binding in each of the sub-pockets reveal trends. Those binding in the adenine sub-pocket tend towards more hydrogen bond donors and acceptors than those in other pockets, as expected. The shapeliness of fragments binding in the various sub-pockets is not quantitatively analyzed, though the interested reader could run these calculations. The 50 most common fragments for each sub-pocket are presented as figures in the supporting information.

 

Aside from extracting interesting cheminformatic trends, what else can you do with these fragments? The researchers took a subset of 624 rule-of-three compliant fragments and recombined them to generate 6,720,637 distinct molecules. The vast majority of these appear to be novel, and among the 218 that had previously been reported in ChEMBL, more than 20% were potent (IC₅₀ ≤ 500 nM) kinase inhibitors.

 

With the inexorable increase in docking speeds, this virtual collection of fragments will be useful for building even larger libraries and using them to find ligands for new kinases. And, as the researchers point out, the collection could be useful for fragment growing or merging to new experimentally identified fragments. This is a resource that should be broadly useful for the community.

From noncovalent fragment to reversible covalent CatS inhibitor

We noted just a couple weeks ago that covalent fragment-based approaches have been on a tear. Much of the recent focus has been on irreversible inhibitors, but as we discussed back in 2013 there is much to be said for reversible covalent molecules too. These are the subject of a new paper in J. Med. Chem. by Markus Schade and colleagues at Grünenthal GmbH.

 

The researchers were interested in cathepsin S (CatS), one of 11 members of a family of cysteine proteases. The enzyme has been implicated in a laundry list of diseases, from arthritis to neuropathic pain to Sjögren’s Syndrome, and indeed a few inhibitors entered clinical trials in the early twenty-first century. However, selectivity turns out to be essential: inhibiting the related cathepsin K can lead to cardiovascular problems and stroke. Molecules that appear selective often  contain a basic nitrogen and so can accumulate in lysosomes, achieving sufficiently high local concentrations to inhibit CatK.

 

CatS is a small (24 kDa) enzyme, ideal for protein-observed NMR. An ¹⁵N-HSQC screen of 1858 noncovalent fragments yielded 18 hits, all of which showed similar chemical shift perturbations (CSPs) suggesting binding in the S2 pocket. X-ray crystallography was successful for three fragments, confirming that they do indeed bind in the S2 pocket. Appealingly, this region of the protein is structurally different from the other cathepsins, suggesting a route to selectivity.

 

The sulfonamide moiety of compound 1 (blue) binds in a very similar fashion to the sulfone of a previously reported reversible covalent inhibitor, compound 16 (red). Growing compound 1 to compound 37 led to a significant boost in potency, and crystallography revealed that the binding mode remained the same.

At this point the researchers sought to remove a few heteroatoms as well as introduce the nitrile warhead from compound 16, yielding compound 39b. Surprisingly, this molecule was no more potent than the non-covalent precursor. However, fragment growing into the S3 pocket yielded a massive boost in potency in the form of compound 44. Further SAR and crystallography revealed that much of the increased affinity is due not to specific interactions in the S3 pocket but rather to a hydrogen bond between the newly introduced amide proton and a main chain carbonyl of CatS. Compound 44 is also highly selective against CatK and CatB, showing negligible inhibition of either at 10 µM. Unfortunately, cellular potencies of representative compounds were down by more than three orders of magnitude, likely due to low permeability.

 

While there is still some way to go to establish whether these molecules will succeed where others have failed, this is nonetheless a nice case of fragment-assisted lead discovery And while one can certainly argue that it would have been possible to derive compound 44 from compound 16 through classical medicinal chemistry, fragments clearly helped.

Poll results: Conferences in the age of COVID-19

Will you go to a conference in person next year? Our latest poll addressed that question and more. The 6-question survey was conducted on Crowdsignal from September 13 to October 31 and was answered by 121 respondents, 116 of which answered all the questions. My personal thoughts on two of the more significant virtual conferences I’ve attended are here and here.

 

The first question, answered by everyone, was “Under what conditions would you attend an in-person conference?”

 

Two-thirds of 119 respondents to the question “Have you attended a virtual conference since the beginning of the pandemic?” had done so, and most of them were satisfied.

 

Most respondents would consider attending a virtual conference, though fewer would consider presenting.

 

 

 

And nearly half of 116 respondents expressed some hesitation to the question, “Are you comfortable having your presentation recorded and viewed later by those who missed the live event?” 

 

 

The last question (“Where do you reside?”) revealed a remarkably diverse group from 21 countries.

 

Overall the main takeaways are that people are comfortable attending and presenting at virtual conferences, but that many are concerned about having presentations recorded and shared. This makes sense: one of the nice aspects of conferences is that you can present preliminary information and discuss emerging (perhaps even half-baked) ideas and ask off-the-wall questions. Still, until our industry comes through with an effective vaccine – or our governments and fellow citizens succeed in reducing transmission – virtual conferences will have to suffice.

 

Finally, on the subject of polling, the US has a rather important election tomorrow. If you are eligible and have not done so yet, please vote. And to everyone else, please wish us luck.