BrazMedChem2010

It really was great to get back to Brazil. Early in November, I attended the 2010 Brazilian Symposium on Medicinal Chemistry that in Ouro Preto. The conference commemorated the work of Carlos Chagas who identified and fully characterized the disease that now bears his name. His work has been described in Wikipedia as “unique in the history of medicine because he was the only researcher so far to describe completely a new infectious disease: its pathogen, vector (Triatominae), host, clinical manifestations and epidemiology”. Chagas worked at and subsequently became the director of the medical research institute founded by Oswaldo Cruz. Although Chagas spent most of his working life in Rio de Janeiro, he was born a Mineiro and only left his home state for his university studies. Ouro Preto is one the ‘must see’ attractions of Minas Gerais (and Brazil) and here are a couple of pictures to give those who were not there an idea of what they were missing.



Although the work of Carlos Chagas is worthy of celebration the current state of treatment of Chagas Disease is most definitely not. Like the better known African Sleeping Sickness, it is a trypanosomal disease and of minimal interest to Big Pharma. The drugs used for treatment have unpleasant side effects and once the disease enters the chronic phase they become a lot less effective. A number of lectures focus on the nature of the disease and I find these particularly interesting, although occasionally gruesome (I always appreciate these reminders of why I never considered pathology as a profession).

Álvaro Romanha delivers an interesting keynote lecture in which he looks back over a long career in parasitology. He describes characterisation of the effects of a number of compounds (including one developed by my former employer) on the Chagas parasite. Lucio Freitas-Junior and Andrei Leitão both talk about cell-based assays which can be used in target identification as well as lead discovery. I hope the folk doing natural product research are taking notes...

Barry Sharpless delivers an entertaining lecture on Click Chemistry and I also enjoy talks by Mike Gelb and Tom von Geldern since these both have a strong medicinal chemistry focus. The work described by Mike used Tipifarnib (1), a farnesyl transferase inhibitor as a starting point. This compound kills T. cruzi and its good pharmacokinetic properties reflect the fact that it had been in clinical development. It turns out that the compound actually inhibits the trypanosomal lanosterol 14-demethylase and this is the reason that it is able to kill the parasite. The optimized compound (2) shows efficacy against acute Chagas in mice and is sufficiently selective not to inhibit human lanosterol 14-demethylase or farnesyl transferase. The focus of Tom’s lecture is African Sleeping Sickness although this is still highly relevant to Chagas Disease. He describes an interesting series of cyclic boronate esters (3), the mode of action of which is still uncertain. If you’re going after Sleeping Sickness you’re going to have to get your drug through the blood brain barrier. Tom describes some of the approaches that the team adopted to optimize pharmacokinetics and achieving good Central Nervous System (CNS) penetration. In the chronic phase of Chagas Disease the parasites take refuge in the cells of their host so the drug has an additional barrier to cross. I wonder how the presence of an intracellular parasite might stimulate expression of efflux transporters in a host cell? That would indeed be sneaky...


Cristiano Guimarães presents analysis of relationships between permeability and physicochemical properties such as polarity and molecular size. However, I am more interested in what he has to say about re-scoring of docking poses. In particular, he notes that conformational entropy lost on binding tends to get over-estimated and has published some of this work. Enthalpy is the focus of the lecture by John Ladbury who describes how calorimetry can be used as a tool for understanding biomolecular interactions and an aid to drug design. The idea is that enthalpy changes associated with binding reflect the extent to which polar interactions form between the molecules in the complex. If you can exploit polar interactions to increase affinity then hopefully you’ll end up in a better place because polarity tends to be associated with better aqueous solubility, selectivity and metabolic stability. However, interpretation of the enthalpy and entropy changes associated with binding remains a challenging problem. I'd suggest taking a look at John's recent publication and recent discussion in the LinkedIn Medicinal Chemisry and Drug Discovery Group if you want to find out more.

That just about wraps up the technical part of this post and there is a page for links to talks. However, I did manage to get a few pictures including one of Carlos and another of his boss at the opening reception.



The following photos were taken at afternoon coffee on the last day of the conference. The first of these shows three of my friends from Rio and it was Daniel who did an excellent job introducing and chairing the molecular design session in which I spoke.



These pictures were taken towards the end of the conference. The paparazzo certainly knows how make the ladies from Porto smile and Carlos does look happy to be passing the baton to Vera who will be organising BrazMedChem2012.



Then it was time for dinner. Tom, Roberto and Ivan were staying in the annex and had to be be summoned. Tom looks a bit hungrier than the other two.



These folk are from FIOCRUZ in Belo Horizonte apart from Claudia (Federal University of Ouro Preto) and Malu who can't resist the photo opportunity. Andrei is looking quite intense in the next photo, Patricia less so.



These two photos are a couple of my favorites. I wonder if Barry is suggesting to John that the adiabatic stereoelectrostatic compressibility of the polarisability tensor may well be the the elusive Universal Efficiency Metric that he is searching for. Of course they could just be swapping fishing stories. One of the highlights of the dinner was Mike playing classical guitar and I was pleased that caipirinha consumption did not interfere with ability to operate a rather bulky digital SLR.



Literature cited

Guimarães & Cardozo, MM-GB/SA Rescoring of Docking Poses in Structure-Based Lead Optimization. J. Chem. Inf. Model. 2008, 48, 958-970 DOI

Ladbury, Klebe & Freire Adding calorimetric data to decision making in lead discovery: a hot tip. Nat. Rev. Drug Discov. 2010, 9, 23-27 DOI

EuroQSAR 2010

I thought this would be a good photo to start the post on EuroQSAR 2010. It really was great fun to be in Rhodes and to catch up with a lot of folk whom I've not seen for a while. The photo was taken at Delphi the day after the conference ended. This the Temple of Apollo where the priestess in residence would inhale hot gases and make predictions... of course nothing like QSAR!



You may well ask what a conference on QSAR has to do with FBDD so I'll try to make the connection clearer. The central problem in QSAR is prediction of affinity so it's a good idea to maintain awareness of the field if you're planning to exploit protein or ligand structures in selection of fragments for screening. Also if you're planning to analyse and design compound libraries or assess druggability then it's useful to know a bit about the molecular descriptors and data analytic methods that form the basis of modern QSAR.

The ultimate goal in QSAR is to start with a molecular structure and predict the physiological effects of the compound. In order to to this you need to be able to predict the extent to which the drug binds to its primary target and a number of anti-targets. You can calculate the extent of binding from the affinity of the drug and its unbound (e.g. to plasma protein) concentration in the vicinity of the target. With typical dosing the unbound drug concentration is a function of both time and location (e.g. intracellular versus extracellular) within the body. If that sounds unbearably complex then I should warn you that it can get a whole lot worse because binding might be slow and you might also need to worry about things like reactive metabolites, isoforms and how toasted the patient got in the pub the night before.

I believe that we're a very long way off seeing this goal achieved. Even when the structure of a protein is known, prediction of affinity for an arbitrary ligand is just not accurate enough. Prediction of unbound concentration at arbitrary location in the body is equally difficult although for some targets it may be sufficient to know the unbound plasma concentration. Nevertheless it is possible to build useful models for some of the pieces in this jigsaw (e.g. binding to plasma protein; IC50 for series of chemically similar receptor antagonists) especially when the process in question is strongly influenced by lipophilicity. QSAR models can be local (i.e. only applicable within a restricted regions of chemical space such as a series of analogs) or global (applicable to any arbitrary molecule). My view is that QSAR models presented as global are frequently ensembles of local models and I have expressed this opinion in print.

I guess that something should be said about the talks after such a long-winded introduction. Some of the speakers have made their lecture slides available. I should first point out that I managed to miss the only talk specifically on FBDD because I was retrieving my camera from my hotel room so that I could photograph a friend doing her poster presentation. The inaugural lecture is given by Hugo Kubinyi and although I saw 'The long road from QSAR to virtual screening' two and a half years ago at an OpenEye meeting in Strasbourg, it is still amusing to see polar surface area and connectivity descriptors cop some flak.

Anthony Nicholls delivers a stimulating lecture entitled 'Information Theory & QSAR'. Nearest neighbor models crop up in more than once in this talk and I liked the suggestion that in validation we should be 'making tests NN resistant'. Nearest neighbours also make an appearance in Han van de Waterbeemd's talk (Assessing Drug Safety and Efficacy through ADME predictions) in the context of 'correction libraries'. The idea behind a correction library is to see if there are systematic errors in the predicted values of a property for near neighbours of the compound for which you're making a prediction. If so the differences between the values measured for the neighbor and predicted for them by the model can be used to correct predictions for new compounds. Of course Orwell would have said that all QSAR models are global...

I enjoyed (not least because he did not appear over-awed by the illustrious inaugural lecturer) the lecture by Alex Tropsha entitled 'Novel Approaches to Chemical Toxicity Prediction Relying on the Entire Structure- in vitro in vivo Data Continuum'. Attempting to sum up the talk in one sentence, I'd say that this was a view of how QSAR modelling might be used to integrate diverse data types that vary in complexity and noise level. One comment that I captured from the 'Notes on chemical descriptors' slide (#14; it's marked 'de-Ku' at bottom left) was that 'descriptors are designed to reflect uniqueness of a molecule in comparison with other molecules'. What does this say about nearest neighbours, I wonder...

Jordi Mestres makes the point in his talk, entitled 'Ligand-based Approaches to In-Silico Pharmacology', that we should STOP using the word 'polypharmacology'. I couldn't agree more although I think the term 'pharmacodynamics' is a couple of orders of magnitude more meaningless. This lecture is about predicting affinity across a range of GPCRs and how these affinity profiles might be used, for example, to anticipate side effects of drugs. Intracellular targets will add complexity because less will be known about unbound concentration of drug in the vicinity of the target.

There's also a session on design of agrochemicals kicked off by Klaus-Juergen Schleifer. All molecular design is subject to constraints and it is always educational to see how people designing molecules for different purposes deal with the constraints that apply to them. Designers of agrochemicals need to deal with different species of plants, fungi and insects in a chemical-unfriendly (e.g. bright sunlight, rain) environment. Pharmaceutical QSAR modellers may find that they learn more in an agrochemical session than a pharmaceutical one.

I must confess to being less than alert on the Friday morning and this may have something to do with over-indulging at the conference dinner and ending up on the beach at 2AM (it seemed a good idea at the time but then it always does). Eric Martin describes how QSAR methodology can be used to integrate affinity and protein structural data for protein kinase inhibitors. Tudor Oprea's lecture (Computer-Aided Drug Re-purposing) is notable because of the use of experimental pharmacokinetic data (this is typically available for marketed drugs) to get a handle on unbound plasma concentration. Aspiring systems biologists, take note.

I've taken a look at some of the talks and it's a good time to summarise before getting on to the fun bit where I share some pictures. Firstly I do not see prediction of affinity for arbitrary molecules (e.g. when there is no measured data for analogs) something we can currently do in a useful and general manner. Secondly, I don't see model validation as a solved problem and a session on the subject is something that the Scientific Committee may wish to think about for EuroQSAR2012 in Vienna. Thirdly remember that, "A theory has only the alternative of being right or wrong. A model has a third possibility: it may be right, but irrelevant" (Manfred Eigen).

The excursion to Lindos provides an excellent opportunity get some pictures. First to be papped is fellow AZ-escapee Han.



I sneak up on Anna (who also doesn't work at AZ any more) as we wait for the bus.



Dick is the man behind CoMFA and Topomers and I've lost count of the number of conferences which we have both attended.



Unfortunately I can't get Yvonne to look at the camera. To her right is Cynthia who co-chaired the session on descriptors in which Yvonne and I both did talks. Andreas and Ylva are smiling because I've told them that the chef has got rotted herring on the menu for the conference dinner.



I finally catch Yvonne as we're waiting for the bus back. I'd not seen Eric since the mid-90s so it's good to get the two of them together in this photo.



David (make sure you're in a country with an almost worthless unit of currency should you presume in his presence otherwise prepare to pay out big time) and Frank have been in this business a long time.



Portraits from the conference dinner.



David is animated but Ant looks in need of a dose of the Poisson-Boltzmanns.



Dimitris appears strangely unconcerned to be in the company of two Transylvanians who are discussing anticoagulant QSVRs (Quantitative Structure Viscosity Relationships) as they admire the tone of his carotid arteries.



Graduate students. Birte (4th from left) did a talk and both Juliana (2nd from left) and Andrea (3rd from left) had their posters selected for oral presentation.



Party animals.

Molecular Interactions

Molecular interactions are an important part of the theoretical framework of modern drug discovery and studying them is a great way to increase your understanding of physicochemical principles of molecular design. One view of molecular design is as a process of tuning the interactions of molecules with the different environments in which they exist. Needless to say, knowledge of molecular interactions is particularly valuable in FBDD. Three Roche scientists have recently published “A Molecular Chemist’s Guide to Molecular Interactions” which should be of interest to anyone working in molecular design and other bloggers ( Derek | Joerg ) have already highlighted the article.

The authors cover plenty of ground and everybody should find their favourite molecular interactions discussed. Given the recent LinkedIn discussion on the value of measuring enthalpy and entropy changes associated with binding, I was pleased to see that the authors noted that interpretation of these quantities is typically difficult. The discussion of cooperativity was useful because we often assume that contributions of interactions to binding are additive.

One of my favorite interactions is halogen bonding and I am pleased to see it discussed in some detail. The halogens all confer a degree of hydrophobicity on a molecule and the heavier halogens (i.e. not fluorine) also exhibit an ability to interact with hydrogen bond acceptors that increases with atomic number. Although this class of interaction is sometimes thought to have been discovered in recent times, it’s actually been around a long time. I can remember learning, as a schoolboy in Port of Spain in the mid-1970s, about why iodine is more soluble in aqueous potassium iodide than in water. I developed this theme a bit more in a light-hearted survey of halogens at EuroCUP in 2008 which starred both Bismarck and a medical writer by the name of Bouchardat who was active in Paris when Pincess Victoria became Queen Victoria. I'm not sure if they ever caught the notorious Parisian dog-poisoner.

Something that I found disappointing was that there was not a lot of information about how much additional affinity you’re likely to get by making the different interactions. This should not be seen as a criticism of the authors who have carried out an impressive trawl of the literature. It’s just disappointing that the information is not available in the current literature base.

I’ve some comments to make on the discussion of hydrogen bonds. There is a widely accepted view that the maximum contribution to affinity that a hydrogen bond between a neutral donor and acceptor can make is just over a log unit and here’s what the Roche authors had to say on the subject:

“Hydrogen bonds always convey specificity to a recognition process but do not always add much binding free energy. Desolvation of the donor and the acceptor must occur for the hydrogen bond to form, such that the effects of hydration and hydrogen bond formation nearly cancel out”

I certainly agree that in some cases the contribution of hydrogen bonds to affinity will be minimal. However, the dataset from which that figure of just over a log unit was derived is actually quite small and not especially diverse in terms of donor-acceptor pairings. I believe that if you can form the hydrogen bond deep in a binding pocket then it can make more than the widely accepted maximum contribution to affinity. We recently published inhibition data which included the example of aza-substitution of a pyridine ring resulting in increases of potency of about two log units against Cathepsins S and L2. Some caution is required in interpreting these results because we didn’t have the relevant crystal structures and the inhibitors are racemic. However, I do believe that these results should make us question the prevailing view of the maximum contribution that a hydrogen bond between a neutral donor and acceptor can make to affinity.

I’ll say some things about the discussion of the hydrogen bonding of sulfonyl groups because it should get you thinking a bit. The authors state that:

“Only 30% of the sulfones and sulfonamides form hydrogen bonds. This raises the question of which type of interaction this functional group prefers.”

I’m not sure that I agree with the second sentence and would be interested to know how many of these sulfones and sulphonamides actually had the opportunity to accept a hydrogen bond. If no hydrogen bond donors are present in a molecule then you can’t really blame the sulfonyl oxygens for making contact with aliphatic carbon in the solid state since that's going to be a better option than getting in the way of the sulfonyl oxygens of a lattice neighbour. Even when a donor is present in the molecule, the favoured interaction may well be with a stronger acceptor than the sulfonyl oxygens.

The authors also took a look at the environments of sulfonyl groups in the PDB and here’s what they had to say.

“Notably, of the sulfonyl groups situated in a hydrophobic environment in the PDB, only 36% are found to interact simultaneously as a hydrogen bond acceptor but 79% of the hydrogen-bonded sulfonyl groups are found to interact simultaneously with a hydrophobic group. These findings clearly indicate a dual character of the weakly polar sulfonyl groups as a hydrogen bond acceptor and as a hydrophobic group.”

I simply don’t buy this idea of sulfonyl oxygens having a dual acceptor-hydrophobic character. Hydrophobicity is a statement of aquous solvation characteristics. It will be easier to place a weak acceptor in a hydrophobic environment than it will to place a strong acceptor there. However, if it’s an acceptor, it’ll still prefer the aqueous environment. Think about the consequences of one of these oxygens accepting a hydrogen bond. When an oxygen atom is hydrogen bonded its ability to accept a second hydrogen bond is likely to be reduced because the donor polarises the acceptor oxygen. Also the second donor will also experience repulsive secondary electrostatic interactions with the existing donor. Provided that the oxygen can still accept a hydrogen bond, it will not be too ‘distressed’ (apologies for anthropomorphising) to be in contact with hydrophobic surface. If you’re interested in this sort of thing then take a look at our article on alkane/water partition coefficients to see how accepting a hydrogen bond (from methanol which we used to model octanol) is likely to affect the ability of carbonyl oxygen to accept a second hydrogen bond.

To be fair, the authors do recognise that accepting a hydrogen bond might affect the probability of a sulfonyl oxygen atom making contact with hydrophobic surface. However, this hydrogen bond donor doesn’t need to come from the protein or a water molecule that the crystallographers can see. How many of the sulfonyl oxygen atoms which lack ‘visible’ hydrogen bonds are sufficiently solvent-exposed to accept hydrogen bonds from ‘invisible’ solvent water molecules? I’ll leave it to you the reader to think about whether analysis of the CSD (as opposed the PDB) has any relevance to hydrophobic interactions.

I’m now going to wrap up with what will be seen by some to be nitpicking although that is not my intention. This is what the authors have to say about QM calculations and hydrogen bonding:

“Where experimental data are not available, acceptor strengths can be obtained from quantum chemical calculations.”

My first criticism of this statement (which is getting very close to nitpicking although as The Blogger I’m allowed to do that) is that quantum chemical calculations can be used to predict donor strengths as well. Like they might say in Buenos Aires, it takes two to tango. My second criticism is that rather than talking about generic “quantum chemical calculations” the authors could also have mentioned that electrostatic potential is a useful predictor of both acceptor and donor strength. I have to declare an interest here as author of reference 98d but I do believe that the effectiveness of electrostatic potential as a predictor of donor and acceptor strength is more important than whether it was calculated quantum mechanically or classically. It tells us something about the nature of the hydrogen bond.

That brings us to the end of my review. The article is definitely a good read and a valuable contribution to the field. To put it bluntly, you need to know this stuff if you want to succeed in FBDD. I’ve flagged up the issue of sulfonyl oxygen hydrogen bonding to hopefully make you think a bit and maybe even generate some discussion. Feel free to make comments of your own.

Literature cited

Bissantz, Kuhn & Stahl, A Medicinal Chemist’s Guide to Molecular Interactions. J. Med. Chem. 2010, 53, 5061-5084 DOI

Davis & Teague, Hydrogen Bonding, Hydrophobic Interactions, and Failure of the Rigid Receptor Hypothesis Angew. Chem. 1999, 38 736-749 DOI

Bethel et al, Design of selective Cathepsin inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 4622-4625 DOI

Toulmin, Wood & Kenny, Toward Prediction of Alkane/Water Partition Coefficients. J. Med. Chem. 2008 51, 3720-3730 DOI

Kenny, Hydrogen bonding, electrostatic potential and molecular design. J. Chem. Inf. Model. 2009, 49, 1234-1244 DOI

LinkedIn Discussion: Enthalpy/entropy & kinetics of binding

Readers of this blog may be interested in a discussion on enthalpy/entropy and kinetics of binding that members of the Medicinal Chemistry and Drug Discovery LinkedIn group are currently having.

A short update

I’m now back in the UK and have been catching up with a few folk. It certainly was a great trip and I’ve set up a travel blog called The Great Escape (better late than never) to share some photos. So far the first month, which included a couple of weeks in Paraguay, has been written up.

You may remember the post on SPR from a while back. It got turned into a letter to the Journal of Molecular Recognition which (to my great amusement) is treated as a publication by Google Scholar. It turned out the review that inspired the post had a few folk spitting feathers and I got the chance to join the fun. I’ve never figured out why people try to eat feathers.

FBDD in Academia 2

Previously I noted that FBDD provides a means for academic groups (and start ups) to negate the advantage that Big Pharma’s massive screening collections give them. Fragment screening and structural characterisation of fragment binding broadens the scope of a structural biology group’s activities. I do believe that a package of fragment binding modes and affinities is something in which a pharmaceutical company would be interested. But what if an academic group wants to move the fragment hits (which I refuse to call ‘frits’ because I don’t work there any more) further along the optimisation trajectory? I’ll outline some of the issues that need to be addressed and this post is intended to stimulate discussion rather than being a last word on academic FBDD. Please feel free to comment if you’d like to challenge anything or flag up anything that’s been overlooked or oversimplified.

The post-screening phase of lead generation will generally require chemical synthesis. Academic synthetic chemists typically focus on synthesising complex natural products or developing new synthetic methodology so it can be difficult to interest them in the more mundane business of lead generation. To be fair the synthetic chemistry required for lead generation is unlikely to be of sufficient novelty or complexity to earn a graduate student the PhD in synthesis which will be his or her primary objective. There are medicinal chemists in academia but in some cases these happen to be synthetic chemists who think that medicinal chemistry is simply a branch of synthetic chemistry. Also synthetic chemists in academia tend not to be interested in molecular design. The net result is that it can be difficult for a protein structure and fragment screening group to find academic collaborators to take the project into the post-screening phase even when there may be synthetic chemists in the same institution.

As you move from screening to post-screening phases of lead generation the work becomes more multi-disciplinary. Unfortunately ‘multi-disciplinary’ isn’t something that usually gets done well in academic institutions although the problems are often less to do with skills than with organisation (and occasionally egos). In addition to the molecular design and synthesis, it will become necessary to run assays to demonstrate that affinity translates into inhibition of the target enzyme (it’s likely to be an enzyme if you’re doing FBDD). For some targets (e.g. antibacterial or kinases) it’ll be necessary to demonstrate some cellular activity. As you approach micromolar potency you may want to check that compounds in your lead series have sufficient aqueous solubility and don’t have particular affinity for anti-targets such as hERG and CYPs. This is much less of an issue if the main objective is publication but is something to be considered if you’re hoping to flog the results of the work to a pharmaceutical company.

That last comment gets me onto a tough issue for an academic lead generation group. How might you persuade a pharmaceutical company to buy your lead series? The main problem is what you’re trying to sell is information and you’ll need to show that you’ve got something good without giving it away. Life is easier if you own the relevant intellectual property but the synthetic chemistry that needs to get done to secure patent cover is not always going to get people PhDs in synthesis. The other point to remember is that Pharma people may not be willing to look at what you’ve got under a confidentiality agreement because of potential for compromising their own IP position. This can become a serious issue if you’re trying to stake a claim for future activity against related targets (e.g. all tyrosine kinases) or to negotiate exclusivity by preventing a company from using leads from competing programs.

So far this post has focussed on the difficulties (which in Pharma-speak would re-branded as personal development opportunities by the happy-smiley folk who inhabit the HR ether) of doing post-screening fragment-based work in an academic environment. If the primary objective is publication then you can write up at any point that is convenient which means that even a small amount of synthesis can have impact. In contrast, commercial lead discovery organisations need to create a secure IP position before they can publish. When publishing affinities and structures of protein-ligand complexes it’s always worth looking out for results that have relevance that goes beyond the specific project. Examples of synthetic elaboration of a fragment leading to a change in its binding mode are particular relevant and the prototypical (low molecular complexity) nature of fragments means that differences in affinity are more easily interpreted.

Getting pharmaceutical companies interested in the output of an academic fragment project is not trivial. A lot depends on the value of the target and the quality of the leads that have been generated. However, getting to leads requires organisation and realising the value of them requires commercial awareness. Organisation is about persuading people that they’re better off working together and can take time to put in place. Commercial awareness is more difficult to acquire and it’s probably best to try to keep things as simple as possible when starting out. Although this may all seem a bit daunting it’s worth remembering that synthesised compounds will typically be novel and that synthesis can be directed away from known ligands. Also one should not forget the ‘supporting data’ of crystal structures and measured affinities.

This is a good point to wrap up. I believe that FBDD provides an excellent framework in which to both train researchers and do high quality science. FBDD also extends the range of options available to academic researchers for collaborating with industrial partners. That's where I'm going to leave it so feel free to comment if anything that I've said (or not said) has annoyed you.

FBDD in Academia 1

I’ve now gone back to being a tourist and will be in Australia until the end of the month before heading north to Singapore and Malaysia for most of June. Feel free to get in touch if you’re based in either of those countries and would like to discuss fragment stuff or Drug Discovery in general.

Some time ago, I promised to post on FBDD in academia and really can’t keep putting this off. I’ve realised that it’s not going to be possible to squeeze everything into a single post so there should be at least one more post after this one. You should be warned that my academic career ended some years before people started to talk about FBDD so if I appear to be out of touch, it may well be because I am out of touch. Hopefully some of what I’m going to say may be of interest to some of you and please remember that this blog does allow its readers to comment.

I’ll start by making two points, both of which will be obvious to many of you. First fragment based approaches provide a means for drug discovery researchers (both in academia and start ups) to counter the advantages that Big Pharma derives from having massive screening libraries and automated compound handling. Secondly measurement of weak binding and determination of binding mode of weakly bound complexes remains a frontier area in physical biochemistry and biophysics. Remember that the power of a binding assay is defined by the weakness of the binding that can be measured reliably.

An academic group with strengths in protein structure determination and biophysical measurement of binding is well placed to contribute. I see the output of protein structural studies moving away from only determining the structure of a protein to providing a more integrated view of the protein’s ‘interaction potential’. One point worth making in this context is that measured thermodynamic parameters for fragment binding are particularly useful for developing and validating theoretical models because there are fewer protein-ligand contacts and it is easier to quantify conformational strain. Fragment based approaches also provide a means to validate and explore bioisosteric relationships without the need for a lot of synthesis and I’ve created a graphic showing how this might work.

Assembling and maintaining a usable screening library is likely to be a challenge or at very least an issue for most academic groups. However, a group that has established expertise in fragment screening does have some advantages in negotiating with suppliers of compounds who may value experimental characterisation of how well their compounds have behave under assay conditions. Vendors of specialist fragment libraries really should value this type of feedback and if they don’t they shouldn’t be in the business of marketing fragment libraries. I sometimes wonder if synthesis of fragments might form the basis for final year undergraduate synthesis projects which could be quite self-contained and include a molecular design component. In passing I’ll pose the question to readers from academia as to whether they think they’ve got molecular design adequately covered in courses at their universities although I’ll have to leave this topic for another post.

As we all know there is more to FBDD than fragment screening. Once you’ve found fragments that bind, tested analogues of these and determined crystal structures, you’ll need to do some synthesis. For a group whose main expertise is characterising binding and protein structure determination this may a good point to bail out and prepare the results for publication. A group with some access to synthesis may wish to take the project a bit further and publish once they’ve observed some SAR. One of the attractions of FBDD for academic researchers is that there are a number of points at which they can choose to write up the project for publication. It is also worth pointing out that FBDD provides an excellent framework to gain understanding of molecular properties and interactions between molecules. This understanding is essential if you’re planning to do molecular design the basis of which is manipulation of these properties with predictable results.

What if academic researchers want to take things further and generate lead series that will be of interest to Pharma? Synthesis will be necessary and life will get more complicated. I’ll pick this up in the next post (Kakadu salties permitting) since there’s quite a bit to say and I’m actually still thinking about this.

Melbourne, FBDD & Facebook

I now have less than a month left in Melbourne and since we’ve just switched over to winter time perhaps the hint should be taken. It has certainly been fun and the project is nicely under control although past experience suggests that it’s usually not a good idea to say that. I’ve not lived in a city since the mid-80s when I was a post-doc in Minneapolis and have really enjoyed the ease of getting around and ready access some of the wide range of music that Melbourne offers. I enjoyed an excellent performance by the ACO Soloists at Hamer Hall and am hoping to return there for a strong dose of Bach in a week’s time. University College, where I’m currently staying, is running a concert series and the second of these promises to be as enjoyable as the first. One of the music tutors at UC plays flute in the VYSO and I got to see them in action last weekend with their truly awesome guest soloist Kana Ohashi. This was also an excellent opportunity to watch the violinists since I was in the third row. The soloist was truly kinetic (difficult to be otherwise with the Tchaikovsky) and the first violin nearest to me appeared to have been given a special 'first violin bob' by her hairdresser. Maybe they will patent it.

On my first trip to the Paris Cat Jazz Club, I found it closed due to flooding (it was the day of The Hailstorm but at least there were back-to-back episodes of Hogan’s Heroes on TV). On returning the following week I was lucky enough to catch Monique diMattina who is an extremely warm, engaging and talented performer. By the way she also writes and composes and, as luck would have it, will be returning there the week AFTER I leave Melbourne. While wandering round town one Saturday afternoon, I caught The Wishing Well on Bourke Street and their next local gig is also the week after I leave. Good reasons to come back, I guess.

Previously, I pointed you towards some LinkedIn groups that are particularly relevant to FBDD. There are also groups on facebook that you might want to take a look at. It’s a bit more difficult to keep discussions going using the facebook groups because you don’t get alerted by email in the same way that you do with LinkedIn. However there are a lot of folk on facebook (especially in universities) and I believe it can play a useful part in extending the FBDD web. Here is a selection of facebook groups that you may find useful:

Fragment Based Drug Discovery (This is the group that is linked to this blog. I do check it frequently and usually respond to queries.)

Crystallography Rocks (Once you’ve got fragments to bind, you’ll want to see how they bind.)

NMR (There are a number of elegant NMR techniques for detection of ligand binding and you’ll find plenty of expertise in this group.)

Chemoinformatics (Particularly relevant to screening library design)

Dan gave my round the world trip a very flattering mention at Practical Fragments which did remind me that I really need to do a post on FBDD in academia since Teddy (who used facebook to tell me where Rapamycin comes from) has also discussed this. As I’m a real sucker for peer pressure, I do promise to make sure that my next blog post focuses on this topic. The FBDD facebook group led to me giving a lecture (I normally call these harangues) in Santiago and through it I’ve also made a couple of contacts in Singapore where I’ll probably do a couple of talks. Being in a facebook group also got me a chance to look round the Australian Synchrotron during maintenance week, when you can get a better look at all the cool stuff. I’ll finish with some pics from that visit.

FBDD and Networking

Reading an account of the session at the ACS on application of computational methods to FBDD, reminded me that it would be a good time to raise awareness of networking groups in this area. Both this blog and Practical Fragments allow readers to comment on posts although this tends not to happen with the frequency that it does at In the Pipeline, probably reflecting the huge readership, frequent updating and diverse content of what I consider to be the best drug discovery blog by a long way.

People interested in FBDD may already belong to a number of relevant LinkedIn groups. The groups offer some advantages over blogs for getting discussions going in that anyone can start a discussion and group members get alerted by email whenever somebody makes a new comment. I’ll list some of these below in case there are some that you’ve not yet heard about.

Fragment Based Drug Discovery (This group is linked by both FBDD blogs)

Label Free Assay Technology Group (It is the assay that makes FBDD possible. The weaker the binding that you can measure reliably, the more powerful your assay)

Structural Biology (X-ray Crystallography, NMR Spectroscopy, Electron Microscopy) (Generally you’re going to need crystal structures to take fragment hits forward)

Job opportunities in Computational Chemistry and Biology, Xray Crystallography, Fragment Based DD

Recently, I submitted the same item for discussion at a number of LinkedIn groups. I invited group members to share their views on the most appropriate technologies for detecting fragment binding. I learned about some new ways to configure SPR experiments and the use of Tm-shift assays. Most of the discussion was in the Structural Biology group (see discussion) although there was helpful input from the relatively new Label Free Assay Technology Group (see discussion) so thank you to all the participants. It was also great to see a couple of familiar faces from my days in Big Pharma, including a co-author from an article that a number of us wrote back in 2007

Interference, PAIN and cysteine pathologies

Dan provided some useful comments on the last post and I think it’s better to respond with a post since this makes everything more visible the readers of both our blogs. I agree with Dan’s point that there are pitfalls, such as compound aggregation, in addition to interference that Adam and colleagues describe in their article. In an ideal situation one would always have the ability to measure weak affinity directly. Protein-detect NMR is one of my personal favorites but you do need labelled protein and, if you want to get full value for your money (labelled protein is not cheap), you’ll also need resonance assignments. The SPR technology is widely applicable and like the protein-detect NMR will provide a direct measurement of affinity (and a whole bunch of other stuff). Isothermal titration calorimetry (ITC) represents another option although I believe that the technique is relatively sample-hungry and more limited than the other two techniques in the weakness of binding that can be measured. Also you do need heat so to speak even though the experiment is isothermal.

Nevertheless you can get to the point of having crystal structures with bound fragments using only a biochemical assay to measure potency. Given that you may well be screening at concentrations one or two orders of magnitude above what is ‘normal’ in HTS, it does make sense to use the approach that Adam and colleagues describe even if you’re going to follow up with SPR or NMR. I do sometimes wonder if the promiscuous behaviour of some inhibitors is due to this sort of interference rather than aggregation. One intriguing question is whether aggregates can ‘inhibit’ by changing spectroscopic and fluorimetric properties of assay mixtures rather than by interacting with proteins. At least there’s usually the option of running assays with added detergent to check for aggregation.

I won’t say much right now about the structural nasties that Jonathan Baell and Georgina Holloway have identified as PAINS since I’ll be visiting Jonathan at WEHI next Friday. I became acquainted with some of these unsavory structural types during my time in Big Pharma and do not believe that their PAINfulness is specific to the AlphaScreen technology that the WEHI researchers are using. Back in those days we had the Decrapper and a program called Flush...

Dan mentioned the Practical Fragments post on a Cruzain Screen so I thought I’d finish with a couple of papers that show how things can get unstuck when you’ve got a catalytic cysteine with a malicious streak. In the dock is none other than PTP1B, a target that is much-loved by disease area strategists and much-hated by screening groups. I’m not going to review the articles or even comment on them right now. Just read them in the correct order and perhaps we can pick up this theme later.

PTP1B: Read this first

PTP1B: Read this second

Literature cited

Baell & Holloway, New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. J. Med. Chem. 2010, ASAP | DOI

Liljebris et al, Synthesis and biological activity of a novel class of pyridazine analogues as non-competitive reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). Bioorg. Med. Chem. 2002, 10, 3197-3122 | DOI

Tjernberg et al, Mechanism of action of pyridazine analogues on protein tyrosine phosphatase 1B (PTP1B). Bioorg. Med. Chem. Lett. 2004, 14, 891-897 | DOI

Interference correction in biochemical assays

Surface Plasmon Resonance (SPR) was in focus recently both here and across at Practical Fragments. However, now I’d like to take a look at using biochemical assays to identifying fragments that bind to targets of interest. Biochemical screens can typically be run in high throughput and are compatible with automation for high throughput screening, which makes it easy to do follow up screening with analogs. Furthermore the hits identified by biochemical assay are actually inhibiting rather than just binding. A criticism of biochemical screens is that they measure binding indirectly and are prone to interference. Sometimes they are used as a pre-screen to reduce the number of compounds that need to be evaluated in a lower throughput biophysical assay. However there are things that you can do to make your biochemical assay more reliable and meaningful. And maybe even more fun.

The article that I’ve chosen to take a look at in this post is by Adam Shapiro and some other colleagues from my days in Big Pharma. Before I met these folk, most of my fragment work had been around libraries for NMR screening and I learned from them how it is possible to correct for some of the interference from test samples in biochemical assays.

Inhibition is typically detected in a biochemical assay by quantifying changes in light absorption, fluorescence or luminescence. In high throughput applications ‘assay components are added serially to wells without any filtration or washing steps’ which means ‘that the test sample remains in the well during the optical measurement and can interfere with it’. This means that compounds that absorb in the UV or visible range and that fluoresce or quench fluorescence can all lead to changes in the readout parameter without actually binding to the target protein. Other less obvious causes of interference include insolubility of test compound (turbidity can lead to detection of highly polarised scattered light) and meniscus deepening which decreases path length. Compounds are typically assayed at relatively high concentrations in fragment screening, making it especially important to recognise and account for assay interference in these applications.

In addition to providing a useful discussion on the causes of interference, the article describes a practical approach to correcting for it by running ‘artefact assays’. These involve running additional plates in which wells contain the same test samples but no target protein. The wells in the artefact assay plate also need to contain whatever is responsible for generating the signal (e.g. reaction product) and a baseline can defined by preparing wells without test samples. The authors describe in some detail how they apply the corrections and since this is only a summary of the article, I’ll leave it to you to go and check their article out. However, I would like to conclude by noting that the authors also suggest criteria for deciding to reject data because interference is too great for meaningful correction.

Literature Cited

Shapiro, Walkup and Keating Correction for Interference by Test Samples in High-Throughput Assays. J. Biomol. Screen. 2009, 14, 1008-1016 | DOI

Surface Plasmon Resonance

General Reviews

Rich & Myszka, Grading the commercial optical biosensor literature – Class of 2008: ‘The Mighty Binders’ J. Mol. Recognit. 2010, 23, 1-64 Link | Review

Application to Fragment Screening

Giannetti, From experimental design to validated hits: A comprehensive walk-through of fragment lead identification using surface plasmon resonance. Methods Enzymol. 2012, 493, 169-218. DOI

Perspicace et al, Fragment-Based Screening Using Surface Plasmon Resonance Technology, J. Biomol. Screen. 2009, 14, 337-349 DOI | Review

Binding Pathologies

Giannetti et al, Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors, J. Med. Chem. 2008, 51, 574-580 DOI | Review

Ligand protein interactions by SPR

I have now been in Melbourne for about a month and have found the city very much to my taste. I’m visiting some friends to help out with some fragment stuff and have already been wreck diving (on the HMAS Canberra) and watched the Australian Open and a rather one-sided ODI between Australia and the West Indies. On the science side of things, I was able to gatecrash Surface Plasmon Resonance (SPR) course, hosted by the Biomolecular Interaction Facility at CSIRO, Parkville, and taught by Rebecca Rich and David Myszka of the University of Utah. Not the whole course, I might add, because the participants spent the second day of the course in the lab and I’m sure there was a clause in my visa agreement that stipulated that I was not to enter a laboratory except as an observer accompanied by a responsible adult.

SPR has always represented a bit of a gap in my knowledge base so this was always going to be a great opportunity. As well as being experts in this field, Rebecca and David present their material with great clarity, enthusiasm, charm and humour. I particularly liked David’s take on the Maxwellian Demon (these molecules don’t have eyes).

When using SPR to screen ligands, the protein is typically immobilised on the surface of the sensor chip. The term ‘immobilised’ is actually a bit of a misnomer and ‘tethered’ would actually be a more appropriate term. The SPR technology can be used to look at diverse types of interaction over a wide range of affinities and kinetic parameters (e.g. on and off rates) can also be measured.

There is of course a slight catch. The experiments need to be performed carefully and this was a recurring theme in the lectures (and presumably in the practical sessions as well). Now it turns out that much of the SPR literature is perhaps based on experiments that have been performed less than perfectly and, as a public service, Rebecca and David have reviewed and graded the SPR literature of 2008. GRADED? Yes, GRADED, and there were some Fs! Of course David is just the person to do the grading since he sports whiskers of which a Victorian (historical context rather than geographical) head master would be justifiably proud and it is easy to imagine him summoning the hapless transgressors to his study.

A grading exercise like this is unlikely to win you many friends and the authors are realistic to accept that it is likely to reduce the likelihood of either being elected to the National Academy of Sciences although hopefully they will never have to employ the services of professional food tasters when they attend SPR conferences. Putting on my computational chemistry hat, I couldn’t help thinking that the QSAR and Virtual Screening fields might benefit from a similar treatment...

There are a number of articles describing the use of SPR to screen fragments against target proteins and the one I’ve chosen to take a look at is from some folk at Roche. One of the authors of this work is David Banner, whose talk at RSC BMCS 2009, I greatly enjoyed, not least because he made no reference to ligand efficiency except, if I recall correctly, to say that he would not be referring to it.

The Roche group screened a library of 2226 compounds against chymase at 200 micromolar and found 80 hits so clearly SPR technology can provide the throughput required to run a fragment screen. The compounds were screened against an inactive (zymogen) form of the protein as a check for non-specific binding. The authors also described cross-competition experiments which could be used to determine whether two fragments were binding at the same or different sites and it is worth remembering that you need to be able to measure binding very directly to get this sort of information. It would have been really interesting if the results of the cross-competition assays had been integrated with crystallography since 12 co-crystallised complexes showed fragments binding in the active site.

Both stoichiometry and kinetics of binding can be determined by SPR making it an appropriate technique with which to observe interactions between badly behaved ligands and proteins. In an excellent (A-graded by Rebecca and David) article, another Roche group exploit SPR to classify some of these binding pathologies. It is particularly good reading for anyone who has worked up results from high throughput screens but that is not a place I particularly want to go to right now since it’s getting rather late at night and I really don’t want to have nightmares about pathological fragments.

Literature cited

Rich & Myszka, Grading the commercial optical biosensor literature – Class of 2008: ‘The Mighty Binders’ J. Mol. Recognit. 2010, 23, 1-64 Link

Perspicace et al, Fragment-Based Screening Using Surface Plasmon Resonance Technology, J. Biomol. Screen. 2009, 14, 337-349 DOI

Giannetti et al, Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors, J. Med. Chem. 2008, 51, 574-580 DOI