Spatial overlap of peptide hotspots and canonical drug pockets in a model enzyme

Spatial overlap of peptide hotspots and canonical drug pockets in a model enzyme was presented by Dr Walraj Gosal, Senior Scientist, Isogenica at the Cresset European User Group Meeting 2015.

Walraj’s talk described the process of moving from peptides from molecular display to small molecule inhibitors, with the help of Cresset technology. In collaboration with Cresset and Biolauncher, the team found that Cresset’s field patterns based on peptides can be used to find new inhibitors. The work was funded by the TSB (Technology Strategy Board).

Molecular (CIS) display1 is an Isogenica technology that allows you to find novel peptides and protein scaffolds that bind a given target.

Walraj described the basic problem: we don’t know how to move from the primary sequence to the precise 3D fold of the protein. He described it as the ultimate needle in a haystack problem, whereby a 100 amino acid protein relates to x 10130 sequences. They are trying to figure out how, if someone comes to us with a target, they can get a sequence that will bind to the target.

Ultimately, the ideal solution would be a complete algorithmic solution and he briefly highlighted recent computational approaches (e.g. Rosetta) that are showing evermore promise towards this goal2,3. However, at the moment the only viable approach is molecular display. For example, Humira – the biggest selling drug worldwide – was one of the first to reach the market that was partially discovered using display technology.

He went on to describe the basic premise of molecular display, which is to have a library of peptides or proteins that maintain their link to RNA or DNA (a ‘genotype to phenotype’ link). The process is then to enrich the library by presenting it with the target over many rounds of selection.

Moving onto the problem the team were trying to solve – can CIS display peptides inform small molecule discovery. Their target choice was thrombin, which Walraj described as ideal for a number of reasons. Firstly, there is already a mountain of medicinal chemistry data available in the public domain due to the race for a direct thrombin inhibitor in Industry. In general, the compounds that have made it on to the market are all based on the substrate, but are very basic – the reasons being that they mimic a key arginine-aspartate salt bridge in the so-called S1 pocket. This led to lead molecules where the bioavailability was low, and clever pro-drug strategies were necessary that eventually led to drugs on the market (e.g. Dabigatran).

Secondly, and more importantly, the team were inspired by the fact that Nature has found alternative solutions to the problem of inhibiting thrombin and Walraj highlighted three: from a tropical bont tick, the mosquito and a medicinal leech4.

So here is the key question: are molecular display peptides going to open up more avenues for drug design, or are they consistent with previous efforts? The answer turns out to be a bit of both.

They found that many of the peptides bound to the active site but some that also bind to an allosteric site – the latter already suggesting that drug design efforts could be focused on other sites largely ignored by Industry. Nevertheless, looking at the active site binders, whilst many of those peptides contained a motif that mimic the natural mosquito inhibitor, most of them appeared unrelated to each other suggesting multiple solutions. A lot of biochemistry was carried out by the team, and eventually the two best peptides were crystallised with thrombin, which confirmed the binding at the active site.

These structures showed orthogonal solutions – one very much based on the Mosquito solution which is to insert a key arginine in S1 in an opposite direction to substrate – incompatible with catalysis. The other solution appeared substrate-like in its path and direction to the S1 site but with a key difference. Here the peptide delivered an extremely novel ‘warhead’ in the S1 pocket that violated the paradigm that the arginine-aspartate salt bridge was required for high affinity. The latter was especially important as the peptide bound with single-digit nanomolar affinity.

They carried out a computational study (using Rosetta) and alanine-scanning mutagenesis experiments (seeing excellent correlation between the two) to determine the key interactions or ‘hotspots’. They then asked whether there was a spatial overlap between the hotspots of these peptides and the canonical drug pockets and interactions that have been exploited over the last 40 years (using data from the PDB). They saw that the hotspots overlap remarkably well with drugs from the PDB. However, some of the high-energy interactions seen in the peptides have never been exploited for drug design. For example, for one of the peptides, a loop movement creates a whole new pocket close to the active site.

Furthermore, the paradigm-violating lipophilic and neutral solution to S1 occupation appears to have been already discovered through HTS and fragment-based design. This highlights the power of molecular display – orthogonal solutions can be discovered remarkably quickly.

The next crucial – and by no means trivial step – is then how you move from these peptide solutions to discovering small molecules? Here, Cresset’s virtual screening technology, Blaze, proved invaluable. The team at Cresset took the crystal structures and produced field patterns based on linear stretches of the peptides incorporating one or more hotspots. For one of the peptides, fewer than 160 of the top compounds suggested by Blaze were experimental screened, and the team found two every small competitive inhibitors of thrombin that were previously unknown.


Walraj concluded that Cresset’s peptide field maps arising from molecular display are sufficient to discover small molecular inhibitors, and combining the power of molecular display and virtual screening would open up a powerful new avenue to drug discovery.

  1. Odegrip, R. et al. PNAS 101, 2806-2810 (2004)
  2. Kuhlman,B. et al. Science 302, 1364-1368 (2003)
  3. Fleishman,S.J. et al. Science 332, 816-821 (2011)
  4. Huntington, J. A. Thromb. Haemost., 111, 583-589 (2014).

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