Pharmacological inhibition of focal segmental glomerulosclerosis‐related, gain of function mutants of TRPC6 channels by semi‐synthetic derivatives of larixol

Abstract

BACKGROUND AND PURPOSEGain of function mutations in TRPC6 channels can cause autosomal dominant forms of focal segmental glomerulosclerosis (FSGS). Validated inhibitors of TRPC6 channels that are biologically active on FSGS-related TRPC6 mutants are eagerly sought. EXPERIMENTAL APPROACHWe synthesized new TRPC6-inhibiting modulators from larixol, a resiniferous constituent of Larix decidua, and tested the potency and selectivity in cell lines stably expressing various TRPC channel isoforms. Channel activation was followed by Ca 2+ influx analyses and electrophysiological recordings. The most promising compound larixyl carbamate (LC) was tested on native TRPC6 channels and TRPC6 constructs carrying FSGS-related point mutations. KEY RESULTSLC exhibited an about 30-fold preference for TRPC6 over TRPC3 channels and a fivefold preference for TRPC6 over TRPC7 channels. Six FSGS-related TRPC6 mutants, including the highly active M132T and R175Q variants, were strongly inhibited by 1 μM LC. Surprisingly, no TRPC6-related Ca 2+ signals were detectable in primary murine podocytes, or in acutely isolated glomeruli. in these preparations. Quantitative PCR revealed a 20-fold to 50-fold lower abundance of TRPC6 transcripts in rat or mouse podocytes, compared with pulmonary artery smooth muscle cells from the same species. Accordingly, electrophysiological recordings demonstrated that DAG-induced currents in murine podocytes are very small, but sensitive to LC. CONCLUSIONS AND IMPLICATIONSIn spite of their low abundance in native podocytes, native TRPC6 channels are targetable using larixol-derived TRPC6 inhibitors. As observed with wild-type TRPC6 channels, FSGS-related TRPC6 mutants were sensitive to the newly developed inhibitors, paving the way for experimental therapies. Abbreviations

A (+)‐Larixol Congener with High Affinity and Subtype Selectivity toward TRPC6

“…Further modifications of the C6 alcoholw ere achieved using the imidazole-activated formate precursor 16,w hich allowed the easy generation of the isosteres 17, 18,a nd SH045.W hile 16, 17,a nd 18 displayed IC 50 valueso f0.44, 0.45, and0 .17 mm, respectively,t he introductiono faN-methylated carbamate functioni nc ompound SH045 yielded a1 5-fold improvement in potency over larixol (1)a nd a3 -fold improvement over the previously described larixyl carbamate 4 [9] with an IC 50 = 63 nm. Furthermore, FLIPR measurements on TRPC3 and TRPC7p rovided ap romising selectivity data for SH045 (IC 50 = 0.84/ 0.22 mm, Figure 3D,E;f or TRPC3 p < 0.05, Figure 4) and therefore outperformed every known congener regarding affinity and selectivity to date.…”

“…[13] While 1 is as ubtype-prevalent blockero f TRPC6 over TRPC3 and TRPC7, epi-manools hows lower affinity and no selectivity,e xplaining the importance of the C6 alcohol function. [8,9] In accordance with previouss tudies, we aimed to expand the knowledge about the structure-activity relationship of 1 by:1 )further diversification of the alcohol at C6, 2) formation of at hird ring structure and oxidation chemistry, and 3) chemical engineering the side chain at C9. [8,9] In accordance with previouss tudies, we aimed to expand the knowledge about the structure-activity relationship of 1 by:1 )further diversification of the alcohol at C6, 2) formation of at hird ring structure and oxidation chemistry, and 3) chemical engineering the side chain at C9.…”

“…Results are referred to IC 50 values in the In previous studies, we reported that potent and subtype-selective TRPC6 inhibitors could be generated by acetylation and carbamylation of (+)-larixol, [8,9] suggesting the high importance of C6 substituents. For all compoundst ested, the calculated logP values range from 4t o7and for some derivatives pluronic F127 wasa dded to increase solubility,w hich had ap ositive effect on bioactivity (see SI section 1.2).…”

“…On the other hand, introducing sterically demanding or rigid elements at position C6 reduced bioactivity in all cases (Figure 4). The observation that an unbranched alkyl chain substituent at this position (n-butyl,a si n 20)w as better tolerated than ab ranched moiety (cyclohexyl as in 21), which is shown by the IC 50 valueso f0 .55 and 1.54 mm on TRPC6, respectively,c learly supports this hypothesis.I nter- www.chemmedchem.org 2018 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim estingly, with an IC 50 = 13.6 mm, xanthate 22,w hichi si solobal to SH045,d id not block Ca 2 + influx as potently.T his shows that small alterations and fine tuning of the electronic properties leads to pronounced effects on bioactivity.P revious studies revealed that ab isacetate is less bioactive than the monoacetylatedl arixol at C6, [9] but the highers ubtypes electivity toward TRPC3 seemed to indicate the benefit of modifying the secondary alcohol. The observation that an unbranched alkyl chain substituent at this position (n-butyl,a si n 20)w as better tolerated than ab ranched moiety (cyclohexyl as in 21), which is shown by the IC 50 valueso f0 .55 and 1.54 mm on TRPC6, respectively,c learly supports this hypothesis.I nter- www.chemmedchem.org 2018 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim estingly, with an IC 50 = 13.6 mm, xanthate 22,w hichi si solobal to SH045,d id not block Ca 2 + influx as potently.T his shows that small alterations and fine tuning of the electronic properties leads to pronounced effects on bioactivity.P revious studies revealed that ab isacetate is less bioactive than the monoacetylatedl arixol at C6, [9] but the highers ubtypes electivity toward TRPC3 seemed to indicate the benefit of modifying the secondary alcohol.…”

“…Recently,t he diterpene (+)-larixol and its acetylated congeners demonstrated selective inhibition of the second-messenger-gated cation channel transient receptor potentialc anonical 6( TRPC6)o ver its close isoforms TRPC3 andT RPC7. [8,9] Spurred by recent computational approaches alongside with biology-oriented synthesis (BIOS), [10] which have ignited ar enaissance in the use of natural products in drug discoverya nd chemical biology,a nd in particular for TRP channel treatment, [11,12] we intended to furthere xplore the larixol scaffold. Implementing high-throughputC a 2 + FLIPR screening assays and electrophysiological patch-clamp recordings, we showcase larixyl N-methylcarbamate, termed SH045,a sacompound with nanomolar affinity and 13-fold subtypes electivity over TRPC3 in stably expressing HEK293 cells.…”