Great things start small: an insight into fragment based drug discovery

Have you ever noticed that great things almost always start small? Take for example Richard Branson, he started the Virgin brand with a student magazine!.

We want ‘big’ things, we have ‘big’ aspirations, we want to jump ‘big’ too soon without thinking that maybe starting from small is the best way to go.

Nowadays, drug development is adopting this thought as well: start small. And this can be achieved by screening for small chemical fragments that bind only weakly to a biological target and only then growing them or combining them to produce a compound with higher affinity. This method is known as fragment-based drug discovery (FBDD) and the main topic of this post.

Think small, think FBDD

Developing a new drug is a complex process. Once the target has been chosen, the process of lead discovery begins. A lead is a compound from a series of related compounds that has some of a desired biological activity. A variety of methods to identify hit molecules exist, but the most commonly used is high-throughput screening (HTS), in which libraries with up to millions of compounds with molecular weights of around 500 daltons are screened and nM binding affinities to target are sought. But could we think ‘small’? Smaller molecules, smaller affinities? Definitely YES.

In contrast with HTS, in FBDD typically about 1,000 fragments are screened using biophysical techniques. The fragments are between 150-250 daltons and mM affinities can be considered useful. After biophysical analysis, candidate fragments are grown to form new interactions using structure-based drug design. Although initial fragment hits have low potency due to their small size, they form high-quality interactions and can be readily optimized into potent lead molecules.

Does size matters then? YES, because the size, complexity and physical properties of small molecules can be more easily controlled than when starting from higher affinity HTS hit.

How can fragment binding be screened?

Biophysical methods to screen fragment binding to its target include NMR spectroscopy, X-ray diffraction, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and mass spectrometry. Which one to use? Each method has advantages and disadvantages in terms of sample consumption, degree of automation and assay complexity, but the general rule is that whatever method you choose, it has to be FAST, EFICIENT, PRECISE and to REDUCE THE NUMBER OF FALSE POSITIVES AND FALSE NEGATIVES. Remember that the number of fragments that are frequently analyzed can go from hundreds to thousands, so it is very important to choose well.

A new effective method for fragment screening

An emerging technique has appeared very recently for fragment screening¹. Microscale thermophoresis (MST) has been broadly applied to investigate biomolecular interaction of a variety of drug targets, thus it seemed perfect for trying it in FBDD… and it is.

In a recent fragment screening made by Sanofi, MST was used in an automated manner to screen a library of fragments targeted against the kinase MEK1 from the MAPK signalling cascade, thus with looks into developing anticancer therapies. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods.

But what I found great about this technology and what marks a difference is related to an important aspect of fragment screening campaigns: The EXCLUSION OF FALSE POSITIVES AND FALSE NEGATIVES. Binding of fragments and small molecules can either stabilize or destabilize target proteins. Destabilization or denaturation of proteins is often accompanied by protein aggregation and MST provides a direct feedback on ligand-induced aggregation and other secondary effects. Thus the information provided by MST prevents false-positive hits from entering later stages of hit expansion, as well as rescuing fragments classified as false negatives.

All in all, this speed technology, used in conjunction with other biophysical techniques has the potential to significantly improve FBDD workflows.

In the early days of FBDD there was some scepticism that it would ever work, for example, “How can such low-affinity compounds be identified?” or “Drug discovery is hard enough starting with a high affinity hit so why go to smaller and lower affinity compounds?”. Today, the consensus has changed. At least nine FBLD projects have led to compounds in phase II or III studies, including one approved drug. The fragment approach has been adopted throughout academia and large pharma and biotech companies, and there are a number of commercially available fragment libraries.

So think about it……… maybe it’s time for you to start small.

“Start small, think big. Don’t worry about too many things at once. Take a handful of simple things to begin with, and then progress to more complex ones. Think about not just tomorrow, but the future. Put a ding in the universe”. Steve Jobs.

1. Linke, P., Amaning, K., Maschberger, M., Vallee, F., Steier, V., Baaske, P., Duhr, S., Breitsprecher, D., Rak, A. An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery. 2015. Journal of Biomolecular Screening. doi:10.1177/1087057115618347