One at a time

Watching biological processes one molecule at a time is an incredibly difficult task… but it is getting easier says Dr Joanna Andrecka, and it could turn drug discovery on its head. 

During the last two decades we have witnessed the birth and subsequent breakneck growth of a new field: single molecule biophysics.

Parallel developments of different techniques created the background for this to happen. Single molecule fluorescence imaging and tracking, optical tweezers, atomic force microscopy, to list only a few, have become the bedrock on which single molecule biophysics is based. This means multiplexed, correlative platforms can probe molecular structure, dynamics and function, unhindered by the averaging inherent in ensemble experiments.

This year, a collaboration between AstraZeneca, biotech instrument maker Lumicks, and the University of Cambridge started that will evaluate the use of dynamic single molecule (DSM) approach. The researchers will use an instrument called the C-trap which combines optical tweezers, confocal microscopy and advanced microfluidics system. This will allow simultaneous visualisation and manipulation of dynamic molecular interactions with sub-pico Newton force, and sub-nanometer spatial resolution.

The aim of the Cambridge project is not to replace the platforms currently used in drug discovery but to complement the existing biophysical technologies and crucially, to identify the key developments needed for successful implementation of DSM technology in the drug discovery process.

Reducing the random…

It is perhaps interesting to note that biophysical technologies have only very recently matured to become key components of drug discovery platforms, with the first structure-based drug design methods using X-ray crystallography employed in the 1990s. It was around this time that single molecule biophysics was born.

The suite of conventional biophysical methods: x-ray crystallography, nuclear magnetic resonance, differential scanning fluorimetry, surface plasmon resonance and calorimetry (to list a selection) enabled drug discovery for more challenging targets. These included protein-protein interactions, binding kinetics – indeed this was then identified as a crucial factor for efficacy and selectivity – and provided the foundation for fragment-based drug discovery.

Biophysical technologies have only very recently matured to become key components of drug discovery platforms 

The concept of rational drug design fundamentally relies on detailed mechanistic characterisation of a target and drug-target interaction. Combination of biophysical methods, protein structural analysis, in vitro assays coupled with cell-based experiments and, ultimately, with metabolic pathways is necessary for improving success rate of clinical trials. While hit generation will most likely remain a random factor in the process, target characterisation and hit-to-lead steps can certainly be improved by introducing new biophysical methods.

One example of a method, which recently had a tremendous impact on drug discovery process, is surface plasmon resonance which provides both, kinetic and thermodynamic parameters previously inaccessible.

Hidden targets

What kind of information can be added by correlative single molecule platforms? The power of these instruments lay in an ability to visualise biological processes with unprecedented temporal resolution, one molecule at the time. The dynamic information is truly unique – it is literally watching molecules in action. Since it is only one molecule at the time, such observations often discover transient states otherwise hidden in the ensemble measurements of other biophysical techniques. These short-lived intermediates are typically crucial for thermodynamic and kinetic aspects of every biological process and might serve as a novel drug target.

Obviously, dynamic single molecule information can dramatically improve our mechanistic understanding of the target at the molecular level. This has been realised by several academic groups worldwide that use and develop single molecule techniques.

It is also logical to look at the changes in target’s behaviour in presence of existing drugs, the underlying mechanism of which is not fully understood. If we manage to further unravel precise molecular basis of diseases and the mechanism of action of the existing leads, we can be more effective in designing the next-generation molecules with very specific and predictable action.

DNA-protein interactions are a good example of where single molecule technologies can offer an exciting opportunity to both validate and improve biological models based on structural information. Protein behaviour can change dramatically upon binding to DNA – from stable binding to very fast translocation, from DNA compaction to DNA unwinding or bending. Crucially for protein function, such dynamic behaviours will depend on binding partners, cofactors and small molecules of potential therapeutic activities. While a structural biology approach aims to capture atomic details of target-drug interaction and other biophysical methods can characterise binding energies and kinetics, dynamic single molecule will visualise real time dynamics of common targets involved in replication, transcription or DNA repair.

Another advantage of single molecule approach is its ability to perform mechanical unfolding experiments with a well-defined reaction coordinate, namely the end-to-end extension between two pulling points. Revealing complex conformational landscape of protein’s native state and being able to identify changes caused by small molecule binding might serve as a new platform for targets such as kinases, GCPRs or disordered protein, otherwise difficult to study.

A change coming?

Can we expect that assays based on single molecule approach will revolutionise the drug discovery process? I hesitate to give a strong statement at this stage, but it is worth remembering that it was single molecule detection that allowed the “impossible” super-resolution revolution in fluorescence-based imaging and development of the next-generation sequencing technologies, changing forever experimental approaches in biology.

Over the years however, some of the techniques remained attributes of highly specialised biophysical labs, where home build instruments with unprecedented detection limits still wait for real biological applications. This situation is now changing. However, we continue to need a vision to unlock the single molecule field by making the technology more accessible to a wider scientific audience and I am certain that with further miniaturisation and automation, we will soon enter an exciting era of breakthrough discoveries in biology and medicine led by single molecule techniques.

The full potential of these tools has yet to be realised. Essential to its successful wider use within the industrial and academic theatres will be the quality of the image and force data analysis, and ultimately, to guide automated systems to change and progress assays in response to results.

The power of the unique Cambridge project is the exchange of knowledge and experience as well as setting up a dialog between academia, high-tech and pharma industry, which should speed up the progress. We shall be prepared to observe rapid and very exciting advances in single molecule science and I believe the best is yet to come.

Author:

Dr Joanna Andrecka is Application Scientist at LUMICKS