Molecular Medicine Tri-Con 2012

Molecular Medicine Tri-Con has just ended in San Francisco, and although it is one of those massive conferences with a very wide scope, from bioinformatics to stem cells, there were some talks relevant to FBLD.

Teddy recently summarized how fragment-based approaches have been used to develop bromodomain inhibitors at GlaxoSmithKline, and they’re certainly not alone: Mark Bunnage of Pfizer described how they used fragment-based methods to discover nanomolar inhibitors of BRD4 that are active in cells. This work was done in collaboration with the Structural Genomics Consortium (SGC), and one well-characterized compound, PFI-1, is being released as a chemical probe to the worldwide research community with no intellectual property entanglements.

There was a heavy emphasis on metrics, and as we saw in the poll last year ligand efficiency and LLE (sometimes also called LipE) seem to be dominant. However, the latter metric is used more for advanced leads than for fragments. Siegfried Reich from the Lilly Biotechnology Center (neé SGX) said of LLE that it is less important where you start than where you end up, though starting from a very polar fragment gives you the luxury of adding lipophilicity during optimization.

Slight changes to fragments can often cause them to bind in different orientations, and the same fragment may bind differently in closely related proteins, but Siegfried argued these multiple orientations could be advantageous by providing multiple opportunities for optimization. Siegfried also mentioned that deconstruction of HTS hits to fragments has been successful at identifying fragments with high ligand-efficiency that could subsequently be optimized to new series.

Richard Law of Evotec gave as clear account as possible of fragment molecular orbital (FMO) calculations, a high-level quantum mechanical method for understanding protein-ligand interactions. Although quantum mechanical calculations are notorious for taking hours, days, or even weeks to run, the calculations can be done much more efficiently by breaking larger molecules into fragments.

Richard also kept thorough notes at a break-out discussion I moderated, and was kind enough to share them; I’ve also made a few additions. There were nine people from both large and small organizations, all but one from industry.

Screening technologies

• As we’ve seen here, SPR seems to be the most common fragment-finding technique; in his presentation, Walter Huber said that it is the primary screening method at Roche.
• "Reverse SPR" - the Graffinity/Novalix technology we’ve discussed previously, has been applied to over 100 targets. It is reversed because the small molecules are immobilised on the chip, in multiple orientations to present different moieties to the target protein. This is also a larger library (~25,000).
• NMR was not being used as much as SPR, and no one at the table was using 19F NMR, though its use does appear to be growing, and compound suppliers are coming out with 19F fragment libraries.
• FCS++ is being used at Evotec; it has the advantage of being high-throughout but still highly sensitive and therefore accommodates a larger than average fragment library (~20,000).

3-D fragments

• There is increasing desire for sp3 (3-dimensional) fragments and a move away from planar fragments, though one participant had seen chemists shy away from too many chiral centers.
• Advantage of more vectors for SBDD, and additional solubility versus otherwise equivalent flat compounds.
• Despite these advantages, often planar fragments yield more hits - likely because planar compounds are more likely to form dispersive/non-specific interactions, whereas sp3-fragments must form very specific H-bond interactions in order to bind. Does sp3 therefore also enrich for enthalpic binders?

Use of indices
LE was used by everyone at the table, whereas LipE/LLE were not used until lead compound stage. BEI/SEI and other metrics were not really used.

FBLD vs HTS
Fragment screening is being used on most programs at many companies, in parallel with HTS and virtual screens. A subset of targets is addressed only with fragment screening either because of target-specific information or specific requirements to lower costs of screening.

Fragment screening of GPCRs
There was very limited experience using FBLD on G-protein coupled receptors, though one company is trying to use nanodiscs to stabilize GPCRs for fragment screening. As more crystal structures are solved people may become more comfortable tackling this target class.

Finally, there was widespread agreement that fragments are ideal for designing in the desired physico-chemical properties of molecules as fragments are developed. The parent fragment is not biasing, and could often be med-chemed away. Richard Law offered the analogy of a rock-climber:

An HTS gets you halfway up the cliff, but the route to the top may not be from where you are, or may just be too difficult to find from that position. Whereas a fragment hit is at the base of cliff but enables you to see and select the exact route to the top that you need.

If you attended the break-out discussion or the meeting please comment on your impressions, and if you missed this conference don’t worry – there are many more great events coming up throughout the year.

Upcoming Webinar on NMR in DD

I wanted to make everyone aware of an upcoming webinar: "You are smarter than you think: Applications of NMR in Hit-to-Lead Discovery". Here are the key learning points:

• Application of NMR in Fragment Screening using both target and ligand based methods
• Utilization of NMR Data, e.g. epitope mapping, in Hit to Lead efforts
• Structure determination of compounds by NMR

What I would love to have from the readers of this blog is suggestions as to what particular topics they feel would be most important to learn about. Obviously, the first two topics are broad and I wanted to give the readers here a chance to help set the specifics that will be discussed. Leave a comment or send me an email directly.

Slow-off, albeit tight, fragments

Practical Fragments recently discussed binding kinetics, and that got me wondering whether any fragments have slow off-rates. Turns out some do: a January 2012 review of protein-ligand energetics and kinetics in Drug Discovery Today by Sara Núñez and colleagues at Abbott summarized a paper published last October in Eur. J. Med. Chem. by Jos Lange et al. In it, Lange and colleagues extensively characterize six inhibitors of the enzyme D-amino acid oxidase (DAAO), a potential target for schizophrenia.

The researchers use biochemical assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to characterize the thermodynamics and kinetics of their inhibitors binding to DAAO. Although all six molecules are fragment-sized, these are not your typical fragments: the weakest has a Kd better than 1 micromolar, and all have ligand efficiencies of 0.79 kcal/mol/atom or better! Three of them are shown below, along with their dissociation constants (determined by ITC) and their dissociation rate constants (determined by SPR).

One interesting aspect of the kinetics is that compound 6 dissociates from the enzyme roughly 50-fold more slowly than compound 3, even though it binds only about 3-fold more tightly. As an interesting aside, compound 1 has a slower on-rate than any of the other molecules, a phenomenon the researchers attribute to tautomerization around the pyrazole.

The researchers go on to measure a number of other properties of these molecules, including Log P, pKa, thermodynamic and kinetic solubility, cell membrane permeability, and in vivo pharmacokinetics. There is a tremendous amount of data here, and it’s a lot of fun to dig into.

With a predicted half-life of more than 2 hours, compound 6 certainly classifies as a slow-off fragment. So is it a better drug? Well, it’s not orally bioavailable, in contrast to some of the other compounds, and it has faster clearance in rodents. Unfortunately the researchers do not report in vivo target modulation, but one has to assume that a schizophrenia drug would need to be oral. Chemists can optimize affinity, thermodynamics, kinetics, and drug-like properties all we want, but the body still has the final say.

Who's Regulating the Regulators?

In two recent ASAP papers in J. Med Chem. Chung et al. from GSK in Stevenage report on their recent efforts in discovering bromodomain inhibitors. Bromodomains are part of the "hot" target class largely lumped together in epigenetic targets. Bromodomains are the sole reader for Acetyl-lysine (AcK) and are thus important components of maintaining the "histone code". These domains are small (~110 aa) and have a common fold of four anti-parallel helices with the peptide recognition site in loops at one end of the helices. There are at least 56 bromodomains encoded in 42 proteins in the human genome.

The GSK group assembled a library that mimicked AcK: compounds had hydrogen bonding functionality and a small alkyl group. 1376 compounds were tested in a fluorescence anisotropy assay. Of these, 132 (~10%) showed >30% displacement of the fluorogenic ligand. After all actives were fully titrated, compounds were soaked into apo crystals. 40 structures were then analyzed by X-ray. The figure below shows how the native peptide is bound, analysis of the crystals showed that the H-bond interaction with the bridging water dominates that with N156, but both influence ligand positioning.
Below the structure of one bromodomain is shown, the yellow spheres are AcK sites. The other figure shows the preferred binding surface and low energy waters involved in AcK mimetic binding. These interacting residues tend to be conserved among bromodomains. The authors conclude the first paper by stating that their fragments are unlikely to discriminate between bromodomains. But, the real question is: is it possible to create compounds from these fragments which can?

And the answer is....[I did say there were two papers] maybe.

People have found compounds which work against bromodomains before (Cpds 1 and 2).
As can be seen they are relatively potent (nM) and decently ligand efficient. Cpd 3 and 4 are fragments found in the screen from GSK. Cpd 3 had ~30 % activity against two bromodomains. The authors admit it was far from the best fragment, but "it was small, efficient, novel, and chemically attractive." Who can argue with that.

Following up with this cpd, they found this compound binds in a manner consistent with its selection: one methyl of 3 mimics the terminal methyl of AcK, the second one overlaps the e-CH2 of AcK, and isoxazole N and O mimic the carbonyl. At 2A resolution, they couldn't differentiate which heteroatom was where, so they made 4 to prove their placement: it was.

As a start to the hit expansion, they used a 3D pharmacophore model to search for commerically avaailable analogs: analog by catalog. The found a series of phenyl-isoxazole with meta-sulfonamides on the phenyl ring. Compound 5a is ~100x more potent than the parent fragment and is very efficient (0.39). The xtal structure of 5a shows that it binds exactly as expected. SAR around this compound showed an increase in affinity, but with the expected loss of LE. This series also suffered from poor solubility. They could not add functionality to the sulfonamide (its pocket is rather hydrophobic), but instead where able to increase solubility through para-phenols (this points towards the solvent). They were able to increase solubility via this route, while keeping potency, but losing LE. This series also had cellular activity.
















This work shows that not all protein-protein interactions are flat and featureless. This work also shows that you can target PPI without having to change the rules: no need to relax the Lipinski Rules. These exciting new results show that the hot new targets in drug discovery play by the same rules as the same old targets.