SBIR-STTR Award

Epigenetic regulation of gene expression via methylation has been implicated in diverse diseases including cancer, diabetes and inflammation, and high throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense drug discovery effort. However, there are significant shortcomings with existing HMT enzyme assay methods, and these are slowing exploration of the therapeutic potential of these emerging targets. Detection of specific methylation events can be quite complicated, and detection of S-adenosylhomocysteine (SAH), the invariant product of all HMT reactions, would be preferred in most cases. However, HMTs are very poor catalysts and many have very low SAM requirements - a combination of factors that creates very stringent sensitivity requirements for SAH-based assay methods. Moreover, direct detection of SAH is a very challenging molecular recognition problem as it requires a reagent capable of discriminating between SAH and S- adenosylmethionine (SAM), which differ by a single methyl group. The available SAH assays - which rely on enzymatic conversion of SAH to a detectable product - are inherently prone to interference from screening compounds and lack the sensitivity needed for detection of some methyltransferases. To overcome this technical gap, we propose to leverage the exquisite selectivity and affinity of naturally occurring SAH-binding RNA aptamers, or 'riboswitches', that control the expression of SAM recycling genes in bacteria. This is a collaborative effort between Dr. Ronald Breaker, who discovered riboswitches in 2002, and BellBrook Labs. Dr. Breaker will use a bioinformatics approach to identify candidate SAH riboswitches with suitable properties from more than 1,000 that are known and characterize the SAH/SAM binding properties of the most promising candidates. BellBrook will incorporate these into a fluorescent SAH sensor suitable for high-throughput screening (HTS) assays and validate it for detection of purified HMTs. This will be the first commercial HTS assay based on an aptamer, and it will overcome the very challenging SAH/SAM discrimination problem with at least 10- fold greater selectivity than has been possible with antibodies. By enabling direct, highly sensitive detection of SAH, the SAH riboswitch sensor (rSen-SAH), will accelerate the screening and profiling of otherwise intractable methyltransferase targets, and thereby make an important contribution to the promising field of epigenetic drug discovery.

Thesaurus Terms:
Affinity;Agreement;Antibodies;Antineoplastic Agents;Aptamer;Area;Assay Development;Bacteria;Base;Binding (Molecular Function);Biochemical;Bioinformatics;Biological Assay;Biology;Catalyst;Cellular Biology;Chemicals;Collaborations;Coupled;Detection;Developmental Biology;Diabetes Mellitus;Discrimination (Psychology);Disease;Dna;Dna Sequence Rearrangement;Drug Candidate;Drug Discovery;Drug Targeting;Energy Transfer;Enzymatic Biochemistry;Enzyme Inhibitor Drugs;Enzyme Inhibitors;Enzymes;Epigenetic Process;Event;Exhibits;Fluorescence Resonance Energy Transfer;Gene Expression Regulation;Genes;High Throughput Screening;Histone Methyltransferase;Improved;In Vitro;Inflammation;Inhibitor/Antagonist;Knowledge Base;Ligands;Malignant Neoplasms;Methods;Methyl Group;Methylation;Methyltransferase;Microbial;Modification;Molecular Biology;Molecular Recognition;Nucleosomes;Oligonucleotides;Peptides;Property;Public Health Relevance;Reaction;Reagent;Recombinants;Recycling;Regulation;Rna Binding;S-Adenosylhomocysteine;S-Adenosylmethionine;Screening;Sensor;Signal Transduction;Stem;Structure;Success;Temperature;Testing;Therapeutic;Thermophilic Organism;Time;Universities;Variant;

Epigenetic regulation of gene expression via methylation has been implicated in diverse diseases including cancer, diabetes and inflammation, and high throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense drug discovery effort. However, there are significant shortcomings with existing HMT enzyme assay methods, and these are slowing exploration of the therapeutic potential of these emerging targets. Detection of specific methylation events can be quite complicated, and detection of S-adenosylhomocysteine (SAH), the invariant product of all HMT reactions, would be preferred in most cases. However, HMTs are very poor catalysts and many have very low SAM requirements - a combination of factors that creates very stringent sensitivity requirements for SAH-based assay methods. Moreover, direct detection of SAH is a very challenging molecular recognition problem as it requires a reagent capable of discriminating between SAH and S-adenosylmethionine (SAM), which differ by a single methyl group. The available SAH assays rely largely on enzymatic conversion of SAH to a detectable product, and are inherently prone to interference from screening compounds and lack the sensitivity needed for detection of some methyltransferases. The lack of suitable assay reagents is delaying and in some cases preventing the screening of potential therapeutic targets. To overcome this technical gap, we are using microbial SAH-sensing RNA aptamers, or "riboswitches", that bind SAH with nanomolar affinity and exquisite selectivity. In Phase I, we established the critical technical feasibility for this approach by showing that SAH binding to a riboswitch can be transduced into fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals without disrupting affinity or selectivity. To achieve this, we split the riboswitch into two halves, such that SAH binding induces assembly of a trimeric complex; this modification vastly improved the sensitivity, selectivity and stability of the signaling. We used the split aptamer assays, called AptaFluor SAH, to detect SAH produced by several HMTs at levels several-fold below the sensitivity limit for current assays. In Phase II we will leverage recent advances in aptamer and nanoparticle technologies to make the novel FP- and TR-FRET based assays suitable for industrial HTS, validate them extensively for inhibitor screening and profiling with HMTs, and establish stability and manufacturing aspects required for commercialization. In addition, we will develop an ultrasensitive ELISA-like assay for detecting HMT activity in biological samples using an innovative split aptamer proximity ligation method. By enabling direct, highly sensitive detection of SAH in homogenous the FP and TR-FRET AptaFluor SAH assay will provide a universal HMT assay platform for inhibitor discovery and lead optimization and allow pursuit of otherwise intractable targets. The solid phase AptaFluor SAH assay will enable discovery of biomarkers and development of companion diagnostic assays for clinical development of HMT targeted therapies. Taken together these developments will accelerate screening of new HMT targets and development of small molecule drugs for cancer, diabetes and other diseases with an epigenetic basis.

Public Health Relevance Statement:


Public Health Relevance:
The regulation of gene expression by chemical modification, called epigenetics, is a promising new area for discovering improved drugs for cancer and other debilitating diseases. We are developing new screening assays for important epigenetic drug targets based on naturally occurring microbial chemical sensing molecules, called riboswitches.

NIH Spending Category:
Biotechnology

Project Terms:
Affinity; Antineoplastic Agents; aptamer; Area; assay development; base; Binding; Biochemical; Biological; Biological Assay; biomarker development; catalyst; Chemicals; Chemistry; Clinical; commercialization; companion diagnostics; Complex; Detection; Development; Diabetes Mellitus; diagnostic assay; Disease; drug discovery; Drug Targeting; Elements; Energy Transfer; Enzyme-Linked Immunosorbent Assay; Enzymes; epigenetic drug; Epigenetic Process; epigenetic regulation; Event; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Freezing; Gene Expression; Gene Expression Regulation; Health; high throughput screening; histone methyltransferase; improved; Inflammation; inhibitor/antagonist; innovation; Lead; Ligation; Malignant Neoplasms; Methods; methyl group; Methylation; Methyltransferase; microbial; Modification; molecular recognition; nanomolar; nanoparticle; novel; Performance; Phase; prevent; Production; Quantum Dots; Reaction; Reagent; Research Personnel; Resort; RNA; S-Adenosylhomocysteine; S-Adenosylmethionine; Sampling; scale up; screening; sensor; Signal Transduction; Site; small molecule; Solid; stability testing; targeted treatment; Technology; Therapeutic; therapeutic target; Time