Inhibitors of aldosterone synthase
Development of selective compounds that exclusively inhibit aldosterone synthesis fir use in therapy of resistant arterial hypertension caused by hyperaldosteronism. Currently there is no drug on the market targeting aldosterone synthase. We are the first to fulfill this gap by solving the crystal structure of aldosterone synthase in complex with substrate and inhibitor. We've identified 4 scaffolds for further development and undergoing the HTS.
According to the latest WHO data, more than 7.5 million deaths worldwide are caused by high blood pressure (hypertension), representing approximately 12.8% of total deaths. The sum of years lost due to the premature death and disability (indicator DALYs) due to hypertension is 58 million. Special attention is given to resistant forms of hypertension (RFH), when the combination of three or more antihypertensive drugs of different classes cannot eliminate the symptoms of hypertension. Patients with RFH constitute 5-30% of patients with hypertension and have more severe symptoms.
To a large extent the progression of RFH is related to the excessive aldosterone synthesis (basic human mineralocorticoid hormone). Aldosterone excess is implicated in the development and progression of several different cardiovascular/renal disease processes, including hypertension, congestive heart failure, chronic kidney disease, coronary artery disease and stroke. High aldosterone concentrations have also been associated with insulin resistance and pancreatic ?-cell dysfunction. Most prominent approach to inhibiting aldosterone-induced cardiovascular injury is to suppress its synthesis (i.e., the inhibition of aldosterone synthase). This strategy prevents the non-genomic actions of aldosterone that are not antagonized by MR blockade. Therefore aldosterone synthase (CYP11B2 or AS) has received growing attention as an attractive therapeutic target for antihypertensive drug discovery.
However, the development of selective and potent CYP11B2 inhibitors is particularly challenging due to high homology to the CYP11B1 isoform (93% amino acid sequence identity with CYP11B2) and other steroidogenic enzymes, especially in the absence of structural information. The progress in AS research was hampered for a long time by the inability to obtain a purified protein due to its low stability and membrane nature and as a result no adequate screening systems available.
We use two alternative approaches in the design of aldosterone synthase inhibitors: 1) screening of more specific inhibitors of the enzyme’s active site (based on our crystal structures of human aldosterone synthase in complex with substrate and inhibitor (PDB ID: 4FDH and 4FDH); 2) screening of peptidomimetics that prevent binding of the redox partner to the mitochondrial P450 (based on our crystal structure of the mitochondrial CYP11A1 in complex with redox partner - adrenodoxin).
For both approaches purified protein expressed in E.coli is used. Protein production is according to our developed and published protocol (Strushkevich N et al. Structural insights into aldosterone synthase substrate specificity and targeted inhibition. Mol Endocrinol. 2013). Availability of purified protein has allowed us to develop and implement a high-throughput screening for testing of potential inhibitors and as a result improve techniques used in other laboratories, e.g. detecting of aldosterone synthase activity in reconstructed system using: a) an enzyme expressed at a low level in eukaryotic cells; b) the radioactive substrate. To obtain a selective inhibitor all experiments will be carried out in tandem with two proteins, CYP11B1 and CYP11B2 (93% homology). In addition, project owners have a number of steroidogenic P450, as well as human liver cytochromes P450, which will be tested for cross-inhibition.
Currently the high- throughput screening of aldosterone synthase inhibitors enabled to identify four different types of compounds with strong inhibitory effect on aldosterone synthase. These compounds will be used for molecular docking using the crystal structure of the complex with fadrazole and for subsequent optimization using small fragments library. A high- throughput screening will be carried out in silico and in vitro using commercial library of chemical compounds of various classes and mechanisms of action (1.5 mln compounds). Obtained hit/s will be used for assessment of inhibition effects in reconstructed system and for IC50 detrmination. We are planning to obtain the crystal structure in complex a hit compound for structure-based drug design and for further optimization.
The design of peptidomimetic will be carried out based on the data about the contact zone of the protein partners, according to the crystal structure of P450 and adrenodoxin. The synthesis will be carried out using an automated peptide synthesizer Intavis utilizing both natural and artificial/modified amino acids. In addition, phage-display technology will be used for the screening of peptides having high affinity for aldosterone synthase.
Our compound will have a high inhibitory activity on a specific target (aldosterone synthase), have a minimal inhibitory effect on other enzymes of the steroid hormones biosynthesis and xenobiotic metabolism, and also have low toxicity.