Strategy for Identifying Repurposed Drugs for the Treatment of Cerebral Cavernous Malformation

Abstract

Background Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans with no approved non-surgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. Methods and Results We developed an unbiased screening platform based on both cellular and animal models of loss-of-function of CCM2. Our discovery strategy consisted of four steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2; a secondary screen of functional changes in endothelial stability in these same cells; a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2; and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2,100 known drugs and bioactive compounds, and identified two candidates for further study, cholecalciferol (Vitamin D3) and tempol (a scavenger of superoxide). Each drug decreased lesion burden in a mouse model of CCM vascular disease by approximately 50%. Conclusions By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

Peripheral Plasma Vitamin D and Non-HDL Cholesterol Reflect the Severity of Cerebral Cavernous Malformation Disease

“…Investigators have rightly cautioned that a biomarker of disease aggressiveness does not necessarily portend a benefit of therapeutic modification, and any effects of vitamin D supplementation in multiple sclerosis remain controversial [32]. Cholecalciferol (vitamin D3) supplement has been shown to decrease lesion burden in a murine model of Ccm2 disease [16]. It is reasonable to postulate whether vitamin D supplementation will be helpful in modulating human CCM disease over time.…”

“…Additionally, the inflammatory cells and their cytokines are modulated by vitamin D [15]. Indeed, cholecalciferol (vitamin D3), identified as potentially disease-modifying by using unbiased target-agnostic screening, was subsequently shown to decrease CCM lesion burden in a murine model of CCM, and to inhibit ROCK activity, known to affect CCM development [16]. Plasma vitamin D level has been associated with a favorable lipid profile related to inflammation and cardiovascular diseases [17], while an inverse association of vitamin D level with CRP has also been reported [15].…”

Combined HMG-COA reductase and prenylation inhibition in treatment of CCM

“…To date, various agents have been tested in preclinical models of CCM, including fasudil, which reduced lesion burden in Ccm1 and Ccm2 heterozygous mice (8), and simvastatin, which stabilized the endothelium of the latter (9) but failed to reduce lesion burden in mice lacking endothelial CCM2 (10), in contrast to cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide) (10). Moreover, fasudil (but not simvastatin) reduced lesion burden and improved survival in sensitized Ccm1 (but not Ccm2) heterozygous mice (11).…”

A New Phenotypic Lexicon for Accelerated Translation

“…The elegant work described in the article by Gibson et al 9 in this issue of Circulation illustrates some of the strategies and experimental tools that might allow drug discovery and translational science to bridge the current gaps. Anchoring their approach in the human genetic evidence that cerebral cavernous malformations (CCMs) are caused by loss of function mutations in the gene CCM2, the authors built a system to allow them to screen thousands of drugs for their ability to rescue this rare anatomic disorder, which many would have considered "undruggable".…”

“…By funneling the original library of 2100 compounds through progressively lower-throughput, but higher-resolution assays, the authors were able to efficiently restrict their in vivo experiments to a limited number of candidates. 2,9 Although this example may represent a fortunate outcome, screens in other systems where complex native context is conserved suggest that modest numbers of drugs need be screened to identify primary hits, presumably because of the number of targets available and the bias toward drug like activity in current bioactive collections. 12 One might imagine, given the safety data available on cholecalciferol, that the regulatory progress of this repurposed compound is unlikely to be impeded by the absence of a specific target, but, for less well-annotated small molecules, there would now be a need for target identification before translation could be considered.…”