Riboswitch Based Methyltransferase HTS Assay for Epigenetic Drug Discovery

DESCRIPTION provided by applicant 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 andquot riboswitchesandquot 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 F rster 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 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