Fragments vs plasmepsins

Plasmodium, which causes malaria, is a nasty bag of tricks. These include the plasmepsins, aspartic proteases that – among other duties – digest the hemoglobin in red blood cells. In a recent paper in J. Med. Chem., an international group of collaborators led by Kristaps Jaudzems and Aigars Jirgensons at the Latvian Institute of Organic Synthesis describe how they discovered inhibitors.

The team started by performing a fragment screen against plasmepsin II (Plm II), one of ten plasmepsins encoded in Plasmodium falciparum. A library of 976 rule-of-three compliant fragments (from ChemBridge) were screened in pools of six using three different NMR methods: STD, WaterLOGSY, and T1ρ. A total of 49 fragments hit at least two assays and were competitive with a known aspartic protease inhibitor, and ten of these showed functional inhibition in an enzymatic assay. Fragment 1 was the most potent.

Crystallography was unsuccessful, but the researchers were able to use ILOE NMR to show that another aromatic fragment could bind near fragment 1. Based on this information, the researchers appended a phenyl moiety to produce compound 3a and obtained a 10-fold boost in potency.

Crystallography still didn’t work, but modeling based on similar compounds on a different aspartic protease suggested that adding another hydrophobic substituent could fill another pocket, leading to compound 3b. At this stage the researchers were finally able to obtain a crystal structure of compound 3b bound to Plm II, which confirmed the predicted binding mode and also revealed another pocket that could be grown into as in in the case of compound 4b. This and several related compounds inhibited the growth of Plasmodium falciparum at low micromolar concentrations and were minimally cytotoxic to mammalian cells.

There is still much to do. Selectivity for the one human aspartic protease tested was generally modest. Also, as the researchers acknowledge, the most active compounds are seriously lipophilic. Still, this is another example of fragment-based lead discovery in academia. More importantly, it provides more ideas on how to tackle a pernicious parasite.

Microscale thermophoresis revisited

One of the less commonly used fragment-finding methods is microscale thermophoresis (MST). This measures the movement of proteins in a temperature gradient; ligand binding changes the movement. When we first described MST in 2012, we noted that the technique seemed relatively low throughput. In a paper recently published in J. Biomol. Screen., Alexey Rak and colleagues at Sanofi teamed up with Dennis Breitsprecher and researchers at NanoTemper (which makes MST instruments) to try to increase this.

The researchers chose the kinase MEK1 and carefully developed assay conditions; their detailed description is a useful resource for those who decide to give MST a try. Adding nonionic detergent to the assay proved to be essential for reproducibility and to prevent the protein from sticking to the capillary or aggregating. Also, rather than relying on the weak chromophores (such as tryptophan) in native proteins, MEK1 was labeled with a fluorescent dye. The substrate ATP was used as a positive control, and the measured affinity was in good agreement with previous results.

The screen itself was performed on a set of 193 fragments that had been computationally preselected as potential ligands for the kinase MEK1 (work we blogged about here). These were serially diluted using automated liquid handling and tested in 12-point dose-response curves to try to determine dissociation constants (Kd values) for each fragment. All together this run of more than 2000 capillary tubes required only 90 micrograms of protein and took less than 7 hours. Retrospective analysis suggested that a single-point screen at 150 µM of each fragment would have caught most of the best hits and cut analysis time to 70 minutes, so it looks like MST is becoming competitive with other biophysical screening methods in terms of time and reagent consumption.

What about results? The overall hit rate was nearly 38%, which is high, though not outrageously so given that the fragments were computationally pre-selected. Of these, the best 25 fragments showed well-defined dose-response curves with
Kd < 200 µM and competition with ATP. One nice feature of the method is that pathological behavior such as aggregation or denaturation could be observed directly in the form of irregular or bumpy MST traces, thus allowing false positives to be rapidly weeded out. Similarly, a loss in fluorescence signal was interpreted as the protein unfolding and sticking to the wells or pipette tips.

It is always useful to cross-check hits in orthogonal assays. As we noted previously, these fragments had previously been screened against MEK1 using surface plasmon resonance (SPR) and differential scanning fluorimetery (DSF). Most of the best hits from DSF were rediscovered by MST, though MST found many hits DSF had missed. In contrast, most of the SPR hits did not confirm in MST. The rank order of hits was also similar for MST and DSF but not for MST and SPR.

A picture is worth a thousand words, and some of the best hits were subjected to crystallography. In fact, 7 of the top 15 MST hits had previously been characterized by crystallography, and 7 new crystal structures could be determined out of 11 additional MST hits for which crystallography was attempted.

Overall then it appears that MST is coming into its own. If you’ve tried it, please share your experiences.

Universal fragments for discovering hot spots and aiding crystallography

A couple years ago we highlighted a paper from Eddy Arnold’s group at Rutgers University in which crystallographic fragment screening revealed over a dozen secondary ligand binding sites on HIV-1 reverse transcriptase (RT). Shockingly, the fragment 4-bromopyrazole bound to every single site, which led us to ask “is this a privileged fragment or a promiscuous binder? And as for the sites with no known functional activity, are these useful?” The Arnold group asked themselves these same questions, and provide answers in a new paper in the open-access journal IUCrJ.

The researchers first considered whether 4-bromopyrazole is special. They collected about 20 halogenated aromatic fragments and soaked these into crystals of HIV-1 RT at concentrations ranging from 20 to 500 mM. Of these, 4-iodopyrazole also bound at multiple sites, but most of the others – even closely related molecules such as 3-bromopyrrole or 4-bromothiazole – did not bind to any.

Next, the authors extended these observations to other proteins. When they soaked their molecules into crystals of the endonuclease from the 2009 pandemic influenza strain, they found that 4-bromopyrazole bound to four sites, including two of three identified in a previous crystallographic fragment screen. In one case, a phenylalanine side chain shifted to open up a new hydrophobic binding site. A similar and previously unobserved shift occurred with a tyrosine side chain when 4-bromopyrazole was soaked into the protein proteinase K. Thus, this fragment is able to identify otherwise cryptic binding sites.

Interestingly, the 4-bromopyrazole binding sites could be strikingly dissimilar, ranging from hydrophobic to mildly electropositive to strongly electronegative. The researchers note that the halogen can form either hydrophobic or polar interactions. Also, one pyrazole nitrogen can act as a hydrogen-bond acceptor while the other can independently act as a donor, and these interactions can be with the protein directly or through bridging water molecules.

Last week we highlighted work from Astex suggesting that secondary binding sites in proteins are common, but in most of those cases the proteins had only one additional site, and only a couple had five or six. In contrast, 4-iodopyrazole bound to 21 sites in HIV-1 RT, although only five of these had sufficiently good electron density to allow the entire fragment to be built. (That is, crystallography only clearly revealed the location of the iodine atom in the others.) How many of these sites are bona fide hot spots, and how many could be predicted using computational techniques such as FTMap?

This is all quite interesting, but, as we asked previously, is it useful? The researchers provide two applications.

First, 4-bromopyrazole may be a general probe to assess whether a protein is ligandable. Soaking crystals of the catalytic core domain of HIV-1 integrase in 500 mM of 4-bromopyrazole revealed no binding sites, in sharp contrast to HIV-1 RT, endonuclease, and proteinase K. Integrase also showed a very low hit rate in a general fragment screen, and a plot of binding sites vs fragment-screening hit rate for three proteins showed a linear correlation. Obviously this is a tiny data set, but if it holds up it could be an easy experimental way to assess the difficulty of targets.

Second, the bromine or iodine atoms in the pyrazole fragments could be used in single-wavelength anomalous dispersion phasing, a useful approach for solving crystal structures. The researchers demonstrated this experimentally for HIV-1 RT, endonuclease, and proteinase K, and suggest that 4-bromopyrazole and 4-iodopyrazole could be inexpensive and helpful additions to a “crystallographer’s toolkit.”

Thus, unlike other frequent-hitters such as PrATs, 4-halopyrazoles might be promiscuous yet specific – and useful. I look forward to seeing whether these "universal fragments" catch on.

Secondary ligand binding sites are common

Anyone who has been exposed to much crystallography will have seen examples where a ligand binds somewhere besides the active site of a protein. This is probably all the more likely in the case of fragments, both because fragments are soaked at high concentrations (and thus weaker ligands can be detected) and also because, being less complex, fragments will be able to bind to more sites. In some cases, such as FPPS and HCV NS3, ligands that bind at these “secondary sites” could be advanced to potent allosteric inhibitors. But how common are such sites? This is the question addressed by Harren Jhoti and Astex colleagues in a paper just published in Proc. Nat. Acad. Sci USA.

The researchers were privileged to have 5590 crystal structures of 24 proteins with at least one bound ligand from crystallographic fragment screens. Careful analysis to exclude buffers and molecules bound at crystallograpic interfaces left them with 53 sites total, with each protein having a fragment bound in at least 1 site; one had 6 (still far from the record 16 sites in HIV-1 RT discussed here). Importantly, 16 of the targets had at least 2 ligand binding sites, with an overall average of 2.2. This number of secondary sites is likely a lower bound, as some sites may have been blocked by crystal packing.

What can be said about these ligand binding sites? The researchers compared the sequence conservation between orthologous proteins from different organisms and found that primary binding sites are more conserved than the overall protein sequences. This is expected because, since the proteins likely have similar functions, there are more evolutionary constraints on the active site residues surrounding the primary sites. Interestingly though, the secondary sites were also significantly conserved, suggesting that they too may have some sort of function.

Protein mobility was also examined computationally, with the thought being that functional binding sites should be more rigid than the overall surface of the protein so as to minimize entropic costs of ligand binding. This turned out to be the case for all primary ligand binding sites, but it was also true for most of the secondary sites. Surprisingly, and in contrast to previous results, there were no differences in normalized B factors (roughly, temperature-related motions) for residues in either primary or secondary binding sites compared with surface residues in general.

Comparing the physical properties of the primary and secondary sites revealed that both were more lipophilic than the rest of the protein surface. Ligands tended to be slightly more buried in primary binding sites than in secondary sites, but there didn’t seem to be any differences among the ligands themselves, though the twelve shown in the paper are mostly “flat.”

These combined results suggest that the majority of proteins have multiple sites capable of binding to small molecule ligands. The researchers note that most of their examples are enzymes, so it may not be fair to extrapolate to other protein classes. That said, many GPCRs also have multiple ligand binding sites.

Secondary binding sites have several things going for them. First, allosteric sites provide a means to target proteins in which the primary binding site is problematic, perhaps because it is too closely related to other proteins. Allosteric sites can also be useful for targeting viral or cancer targets in which resistance is an issue, as in the case of ABL001. Finally, secondary sites provide an opportunity to develop not just inhibitors, but activators.

Of course, just because a fragment binds at a site doesn’t necessarily mean that the site is ligandable. Indeed, HSP70 appears to have 5 sites, yet by all accounts is an extremely difficult target. Four of the proteins (including HSP70) are described in some detail in the paper, with protein-fragment structures deposited in the protein data bank. It would be interesting to see how the secondary sites score as potential hot spots using software such as FTMap.

Still, knowing that secondary binding sites are the norm rather than the exception gives new impetus to look for them. It also suggests new areas of biology to explore. Molecular complexity is one thing, but it pales in comparison to biological complexity.

Fragment events in 2016

Happy 2016! This looks like a good year for fragment events, so start planning now!

February 21-24: Zing conferences is holding its inaugural Structure Based Drug Design Conference in Carlsbad, California. This looks like a cousin of their 2014 Caribbean meeting, so it should be interesting.

April 20-21: CHI’s Eleventh Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of last year's 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.

June 7-10: Although not strictly fragment-focused, the third NovAliX Conference on Biophysics in Drug Discovery is likely to have lots of relevant talks, and is a good excuse to get to Strasbourg, France. You can read Teddy's impressions of the 2013 event here, here, and here.

July 12-15: The second FBDD Down Under will be held at Monash University in Melbourne. The first was lots of fun (see here) and even resulted in a special issue of the Aust. J. Chem., so definitely check this out if you can.

October 9-12: FBLD 2016 will be held in Boston, MA. This marks the sixth in an illustrious series of conferences organized by scientists for scientists, the last of which was in Basel in 2014. Surprisingly, this also seems to be the first dedicated fragment conference in Boston. You can read impressions of FBLD 2012, FBLD 2010, and FBLD 2009.

November 7-9: Finally, the OMICS Group is holding their second Drug Discovery & Designing in Istanbul, Turkey, with a track on FBDD.

Know of anything else? Add it to the comments or let us know!