Biophysics in the Alsace

Two weeks ago, the first Novalix conference on Biophysics in Drug Discovery was held in Strasbourg.  I was lucky enough to be one of 160 people in attendance (this was largely a european affair, with ~10% of attendees from outside the EU).  The split of attendees was 60/40 industry/academia.  The conference was split into four themed sessions: Biophysical characterization, Mechanistic Analysis, Emerging Technologies, and Biophysical Methods for Identifying Hits and Leads.  This was not a fragment conference, but many of the talks were specifically about fragments, and the rest could be impactful in fragments.  I want to share my impressions/thoughts on the speakers relevant to the readers here.  You can also go to my website to see my thoughts on the speakers not relevant to FBHG. 

Michael Hennig- Roche: His talk discussed the various methods and showed examples for each.  This was a great talk giving a great overview of the various methods available for active follow up.  Specifically fragments: The Roche fragment library is ~5000 compounds.  In terms of QC, 80% of the samples show >85% purity (by LC-UV-MS).  Purity of fragment libraries has been discussed here previously.  For Roche's uses, every fragment hit is followed up by MC and NMR, so a lower threshold of purity will not have a negative impact.  He also presented results from a 2D-HTS.  This was a new concept for me and I found it intriguing.  The basic concept is to graph the results from two screens (or related proteins) to identify compounds that activate one, but not the other, or activate one and stimulate the other, etc.  He also presented direct and in-direct methods using Mass Spec methods.  To me, this area was one of the more fascinating areas discussed at the conference.  Theoretically, this could be applicable to fragments, but I would really like to see specific applications.  Lastly, he spoke on biophysical methods and membrane proteins. 

Rob Cooke- Heptares:  He presented the STaR approach that has been widely published and presented here, here, here, and here.  The talk was very similar to other talks that Heptares and Rob have presented in various fora over the past year.  The main thing that I was taken by was that there was no mention of NMR.

Matthias Frech - Merck: I really enjoyed this talk.  One of the main things I noted was his use of the phrase "hit affirmation".  Confirmation (according to the dictionary) is a piece of corroboration, while affirmation means it is true.  Is this parsing meaning where none exists?  Maybe, but I think it may also inform on mindset.  He said that SPR is the workhorse for FBHG, but they also use NMR, MST, ITC, stop-flow, and X-Ray.  95-98% of their projects are accomplished using SPR and ITC.  However, he stated that SPR is used to rule out compounds, not rule them in.  This is key to the proper use of SPR.  I would be interested to see if anyone else takes this approach.  They use SPR and ITC to obtain the enthalpic and entropic terms for compound binding.  ITC yields the enthalpy, SPR yields the DeltaG et voila, simple math (my favorite kind) yields the entropic term.  One other very interesting item that he noted was that there was no correlation between affirmation rate and target class for 31 projects they undertook (2009-2012).  

 

Tomorrow I will update the Mechanistic Analysis session.  

Biophysics in Drug Discovery

Folks,

Posting has been light since Dan has been traveling and I have been super busy.  The fun news is that I will be attending the Novalix conference on Biophysics in Drug Discovery this week.  The agenda is shaping up to be very exciting and I think of keen interest to the readership of this blog.  I will (WiFi-willing) be blogging daily about the conference and tweeting random thoughts about the talks. 

For those of you who will be there, I am always happy to meet up and chat.  There are a number of very interesting Fragment-focused talks, so I will be focusing on those. 

More techniques: NovAliX and Graffinity combine MS and SPR

We’ve written previously about Graffinity, which uses surface plasmon resonance (SPR) to find fragments, and NovAliX, which initially focused on mass spectrometry. The two companies have been collaborating since last year, and this has apparently been a fruitful partnership: this month NovAlix acquired a majority ownership stake in Graffinity (click here for press release). Earlier this year NovAlix also purchased an NMR-focused company, and they have already added crystallography expertise. Finding fragments effectively requires using a range of orthogonal technologies, and this latest move gives NovAlix a full suite of biophysical techniques.

Native Mass Spectrometry

We’ve recently blogged on the biophysical techniques surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). This post discusses native mass spectrometry (MS), which was reviewed in Future Med Chem earlier this year. (Thanks to author Denis Zeyer for pointing this out).

Mass spectrometry involves ionizing a molecule, measuring its mass-to-charge ratio, and using that ratio to determine molecular weight. Since the process occurs in a vacuum under high electric fields, biomolecules such as proteins are usually denatured. However, under careful conditions, not only can biomolecules be kept in their native state, but complexes of multiple molecules can be kept together; by measuring the weights of these complexes, the individual components can be determined.

In the case of protein-small molecule complexes, the technique can be used to determine binding and stoichiometry (how many small molecules are bound to a given protein), and the authors discuss a number of papers in the field (including some seminal RNA-fragment examples).

Two of the authors are applying native MS to fragments at French company NovAliX. They describe screening their 350-compound fragment library against the anticancer target Hsp90 to identify 40 fragments that bind to the protein, and further characterized some of these crystallographically. The entire screen, in duplicate, required 2 milligrams of protein.

There are a few limiting issues with native mass spectrometry. First, the technique requires careful choice of buffers; in particular, detergents are not compatible. That can be a problem because omitting detergents sometimes leads to small-molecule aggregation, even with legitimate binders. The authors note that multiple binding is sometimes observed in native mass-spectrometry; it would be interesting to follow up on these observations with activity assays to determine how often these are truly non-specific or just appear so under the assay conditions.

Another issue is that the stability of protein-small molecule complexes in native mass spectrometry assays does not necessarily correlate with the (more relevant) solution-phase affinity. In the gas phase, polar interactions such as hydrogen bonds and electrostatic interactions are strengthened, while the hydrophobic effect is weakened. Intriguingly, a window into gas-phase affinity could actually be an advantage for fragment-based approaches. Polar interactions tend to be enthapically driven, while hydrophobic interactions contribute to overall affinity primarily through entropic effects. If it is true that fragments showing predominantly enthalpic binding are more attractive starting points than those whose major binding energy comes from entropy (as argued here), mass spectrometry may be a good way of finding these fragments. I don’t recall seeing a systematic study dissecting the free energies of binding of hits from native mass spectrometry into their enthalpic and entropic components. If you know of one, I would be interested to hear about it in the comments section.