Facilitating innovation - A robust and reliable platform for early-stage drug discovery and tool compound development
Lead Research Organisation:
University of Leeds
Department Name: Sch of Molecular & Cellular Biology
Abstract
Our organisms are powered by biochemical reactions from molecular machines often made up of individual proteins or protein complexes. Proteins are responsible for tens of thousands if not millions of molecular interactions either with each other (protein-protein interactions) or with small molecule ligands, metabolites and drugs (protein-ligand interactions). Measuring these interactions precisely and in high-throughput is a major challenge and forms the basis for our understanding of how biological molecular machines work and for imagining new ways to develop therapeutic drugs to treat diseases.
We would acquire a Dianthus instrument that can precisely and robustly measure protein-protein and protein-ligand interactions in solution where optimal conditions for protein stability can be maintained. The instrument can simultaneously use two detection modes based on two different biophysical techniques: a spectral shift technology and a temperature related intensity change (TRIC) technology. Each has its own advantages, and the combination is really powerful in improving detection sensitivity (allowing us to use less of the precious materials we make) and can signpost potential "bad" reactions that need repeating or are untrustworthy.
Doing experiments in a high-throughput format with a 384-well plate measuring >1000 reactions per hour will significantly accelerate our ability to discover new molecules as tools to understand biology and disease conditions. It will transform the way we discover and optimise drug candidates for early-stage drug discovery programs. We use multiple screening platforms to identify drug candidates and have a combination of techniques and expertise to optimise "hit" compounds. However, we lack a technology that can provide affinity measurements and rank our molecules according to their ability to bind or "stick" to our biological targets. The Dianthus instrument will bridge this crucial, informative gap in our discovery pipeline.
Many of our projects will benefit from this technology. New enzyme families and drug candidates in the ubiquitin proteasome system promise to benefit inflammatory diseases (e.g. Lupus, Sclerodema, Rheumatoid Arthritis) and multiple types of cancer. These are hard targets because as large multimeric complexes they are difficult to produce and assay with current technology. Similarly, self-assembling systems that form protein fibrils and contribute to amyloidosis in Alzheimer's and type II diabetes are hard to study with current techniques. The new instrument will make it easier to discover molecules that prevent fibril formation. We have teams working on challenging targets that control protein folding and energy flow in mitochondria with promising molecules that may benefit patients with an aggressive and hard-to-treat brain cancer (glioblastoma). We made huge progress with membrane proteins that are targets for malaria, antibiotic resistance and heart disease and are developing new ways to drug these proteins. The 384-well plate system is also ideal for a ligand-discovery platform developed in Leeds which also performs chemical reactions in 384-well plates, ensuring perfect compatibility.
Finally, we are using Dianthus to understand fundamental biology by measuring interactions of protein complexes with DNA to study how DNA damage is repaired. This will further our knowledge of disease biology and help us unveil a cell's "Achilles hill" to unlock new therapeutic targets in cancer.
At time of writing, no UK institution has installed this technology. As such, the Dianthus instrument will be an amazing unique addition to our multiuser research facilities and complement our current capabilities. The instrument will also be hugely beneficial to the Leeds science ecosystem and local universities through our equipment sharing portal for northern universities and other interested institutions.
We would acquire a Dianthus instrument that can precisely and robustly measure protein-protein and protein-ligand interactions in solution where optimal conditions for protein stability can be maintained. The instrument can simultaneously use two detection modes based on two different biophysical techniques: a spectral shift technology and a temperature related intensity change (TRIC) technology. Each has its own advantages, and the combination is really powerful in improving detection sensitivity (allowing us to use less of the precious materials we make) and can signpost potential "bad" reactions that need repeating or are untrustworthy.
Doing experiments in a high-throughput format with a 384-well plate measuring >1000 reactions per hour will significantly accelerate our ability to discover new molecules as tools to understand biology and disease conditions. It will transform the way we discover and optimise drug candidates for early-stage drug discovery programs. We use multiple screening platforms to identify drug candidates and have a combination of techniques and expertise to optimise "hit" compounds. However, we lack a technology that can provide affinity measurements and rank our molecules according to their ability to bind or "stick" to our biological targets. The Dianthus instrument will bridge this crucial, informative gap in our discovery pipeline.
Many of our projects will benefit from this technology. New enzyme families and drug candidates in the ubiquitin proteasome system promise to benefit inflammatory diseases (e.g. Lupus, Sclerodema, Rheumatoid Arthritis) and multiple types of cancer. These are hard targets because as large multimeric complexes they are difficult to produce and assay with current technology. Similarly, self-assembling systems that form protein fibrils and contribute to amyloidosis in Alzheimer's and type II diabetes are hard to study with current techniques. The new instrument will make it easier to discover molecules that prevent fibril formation. We have teams working on challenging targets that control protein folding and energy flow in mitochondria with promising molecules that may benefit patients with an aggressive and hard-to-treat brain cancer (glioblastoma). We made huge progress with membrane proteins that are targets for malaria, antibiotic resistance and heart disease and are developing new ways to drug these proteins. The 384-well plate system is also ideal for a ligand-discovery platform developed in Leeds which also performs chemical reactions in 384-well plates, ensuring perfect compatibility.
Finally, we are using Dianthus to understand fundamental biology by measuring interactions of protein complexes with DNA to study how DNA damage is repaired. This will further our knowledge of disease biology and help us unveil a cell's "Achilles hill" to unlock new therapeutic targets in cancer.
At time of writing, no UK institution has installed this technology. As such, the Dianthus instrument will be an amazing unique addition to our multiuser research facilities and complement our current capabilities. The instrument will also be hugely beneficial to the Leeds science ecosystem and local universities through our equipment sharing portal for northern universities and other interested institutions.
Technical Summary
The Dianthus instrument offers a plate-based and microfluidics-free affinity screening platform. Measurements are in solution and mass-independent, ideal for studying challenging drug targets such as multiprotein complexes, aggregation-prone proteins and membrane proteins. Moreover, the technology was optimised for measuring binding affinities of molecular glues (including PROTACs) and covalent inhibitors, which are useful molecular probes to understand biology and are advancing many therapeutic areas.
Dianthus combines two biophysical techniques: Spectral Shift and Temperature Related Intensity Change or TRIC (see proposal for details). The technology requires no immobilisation, allows screening of a broad range of ligand concentrations (pM-mM) and is compatible with additives, detergents, DMSO, cell lysates and aggregated or heterogenous systems. Two detection modes in one instrument allows measurement of multiple types of interactions, including unstable proteins that require specific buffers & detergents, and interactions between ligands and large, dynamic protein complexes. This is timely as we continue to study more complex and challenging biological targets.
The methodology can determine binding constants of classic protein-protein, protein-ligand (e.g., enzyme substrates, nanobodies/Affimers, nucleic acids) and RNA-ligand interactions, and will be a useful resource for the academic community beyond the applicants in this proposal.
Benefits of proposed equipment:
1. Data with higher signal-to-noise ratio (than MST) - higher confidence in results and can measure high affinity interactions at low concentrations
2. Robustness against aggregates, impurities or buffer conditions
3. Isothermal measurement - suited for temperature-sensitive or labile molecules
4. High throughput (384-well plates) and fast measurement - 16-point Kd in less than 30 seconds
5. Low reagent requirements (15-25 microlitres/well), solution-based assay
Dianthus combines two biophysical techniques: Spectral Shift and Temperature Related Intensity Change or TRIC (see proposal for details). The technology requires no immobilisation, allows screening of a broad range of ligand concentrations (pM-mM) and is compatible with additives, detergents, DMSO, cell lysates and aggregated or heterogenous systems. Two detection modes in one instrument allows measurement of multiple types of interactions, including unstable proteins that require specific buffers & detergents, and interactions between ligands and large, dynamic protein complexes. This is timely as we continue to study more complex and challenging biological targets.
The methodology can determine binding constants of classic protein-protein, protein-ligand (e.g., enzyme substrates, nanobodies/Affimers, nucleic acids) and RNA-ligand interactions, and will be a useful resource for the academic community beyond the applicants in this proposal.
Benefits of proposed equipment:
1. Data with higher signal-to-noise ratio (than MST) - higher confidence in results and can measure high affinity interactions at low concentrations
2. Robustness against aggregates, impurities or buffer conditions
3. Isothermal measurement - suited for temperature-sensitive or labile molecules
4. High throughput (384-well plates) and fast measurement - 16-point Kd in less than 30 seconds
5. Low reagent requirements (15-25 microlitres/well), solution-based assay
