Santalol Isomers Inhibit Transthyretin Amyloidogenesis and Associated Pathologies in Caenorhabditis elegans

Transthyretin (TTR) is a homotetrameric protein found in human serum and is implicated in fatal inherited amyloidoses. Destabilization of native TTR confirmation resulting from mutation, environmental changes, and aging causes polymerization and amyloid fibril formation. Although several small molecules have been reported to stabilize the native state and inhibit TTR aggregation, prolonged use can cause serious side effects. Therefore, pharmacologically enhancing the degradation of TTR aggregates and kinetically stabilizing the native tetrameric structure with bioactive molecule(s) could be a viable therapeutic strategy to hinder the advancement of TTR amyloidoses. In this context, here we demonstrated α- and β-santalol, natural sesquiterpenes from sandalwood, as a potent TTR aggregation inhibitor and native state stabilizer using combined in vitro, in silico, and in vivo experiments. We found that α- and β-santalol synergize to reduce wild-type (WT) and Val30Met (V30M) mutant TTR aggregates in novel C. elegans strains expressing TTR fragments fused with a green fluorescent protein in body wall muscle cells. α- and β-Santalol extend the lifespan and healthspan of C. elegans strains carrying TTRWT::EGFP and TTRV30M::EGFP transgene by activating the SKN-1/Nrf2, autophagy, and proteasome. Moreover, α- and β-santalol directly interacted with TTR and reduced the flexibility of the thyroxine-binding cavity and homotetramer interface, which in turn increases stability and prevents the dissociation of the TTR tetramer. These data indicate that α- and β-santalol are the strong natural therapeutic intervention against TTR-associated amyloid diseases.


Isolation of santalol isomers
The East Indian sandalwood oil was extracted from the heart-wood of plantationgrown Santalum album L. (Santalaceae) trees under Good Manufacturing Practice (GMP)/Good Laboratory Practice (GLP) regulatory guidelines was obtained from Quintis Forestry (Australia) Pty Ltd., (Perth, Western Australia). A second distillation under vacuum (rectification) was conducted to remove low-boiling santalenes that are typically present at low levels in sandalwood oil. The santalol isomers were purified from sandalwood oil by sequential column chromatography on silica gel (Daramwar et al., 2012), followed by supercritical fluid chromatography on chiral support (Averica Discovery Services, Worcester, MA, USA). The purity of isolated santalol isomers was determined by various spectral and analytical techniques, including gas chromatography-flame ionization detector (GC-FID), fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance ( 1 H, 13 C, and 2D NMR), liquid chromatography-mass spectrometry (LC-MS), and elemental analysis.

C. elegans strain maintenance and age synchronization
All the worm strains were maintained and propagated onto the nematode growth media (NGM) agar plates (17 g agar, 2.5 g casein peptone, 3 g NaCl, 1 mL of 1 M CaCl 2 , 1 mL of 1 M MgSO 4 , 1 mL of cholesterol [5 mg/mL], 1 mL nystatin [10 mg/ml], 25 mL potassium phosphate buffer, and distilled water) seeded with live E. coli OP50. For age synchronization, gravid adult worms were collected from NGM plates, washed thrice, and the worms pellet was resuspended in 3.5 mL of M9 buffer (6g Na 2 HPO 4 , 3 g KH 2 PO 4 , 5 g NaCl, 0.25 g MgSO 4 •7H 2 O, and distilled water). Afterward, freshly prepared 0.5 mL 5 N NaOH and 1 mL of household bleach (sodium hypochlorite/ NaOCl) were added to the worm suspension and vortexed for 6-10 min until the bodies of the entire worms dissolved. The eggs were then washed 4-5 times with M9 buffer to completely remove traces of NaOH and bleach. After the final wash, eggs were resuspended in M9 buffer and incubated at 20°C to favor the hatching (Brenner, 1974;Stiernagle, 2006

Food sensing behavior (basal slowing response to food)
Assay plates were prepared in a 9 cm diameter Petri dish by spreading E. coli OP50 overnight in a ring with an inner diameter of ~1 cm and an outer diameter of ~8 cm on NGM agar. After treatment with santalol isomers, the nematodes (n=30-40 per treatment) were washed with M9 buffer and released to the center of the NGM agar plate spotted with or without E. coli OP50 lawn. After five minutes, the body bends of each nematode were measured for 1 min in the presence or absence of food, and the slowing response was calculated using the following formula; Slowing rate = (N without food -N with food ) / N without food Where N represents the total number of body bends in the presence or absence of a bacterial food source.

Chemotaxis assay
Chemotaxis assay was performed according to Bargmann et al., method (Bargmann et al., 1993). Briefly, the age synchronized worms (n=100-120 worms per treatment) were treated with α-and β-santalol. Day 5 and day 10 adulthood stage worms were transferred to chemotaxis plates divided into four equal quadrants (A1, B1, A2, B2) carrying 10 µL attractant (1 M sodium acetate) on one side (A1, A2) and 10 mL distilled water on the other side (B1, B2). 25 mM sodium azide was spotted on each side to paralyze the attracted worms towards the region. The worms were released at the center of the plates and incubated at 20°C for 90 min, and the chemotaxis index (CI) was calculated using the following formula; Chemotaxis index (CI) = (A1 + A2) -(B1 + B2) / N Where A1 and A2 represent worms in the attractant region, B1 and B2 represent worms in the control region, and N represents the total number of worms. Three independent trials were performed with appropriate replicates.

Measurement of body bends
Control and treated worms were washed twice with M9 buffer and released onto the unseeded NGM plates to crawl for 5 min. Individual worms were then transferred to 24-well microtiter plates containing 1 mL of M9 buffer. After 1 min recovery period, the number of body bends was scored for 30 seconds using a stereo zoom microscope. The reciprocating motion of bending at the mid-body of C. elegans was considered as body bend.

Determination of the antioxidant enzyme activities
Worms were treated as said in the lifespan assay. After treatment, control and treated worms (500 individuals per experiment) were homogenized, and the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured according to the instructions provided by the manufacturer. Total protein content was determined by the BCA assay kit, and the results were normalized by total protein contents represented as U/mg protein or nmol/mg protein.

Molecular modeling
Molecular dynamics simulation was performed with GROMACS 2016.4 (Hess et al., 2008) with a CHARMM36 force field and TIP3P water model (Jorgensen et al., 1983;Vanommeslaeghe et al., 2009). Periodic boundary conditions were applied in three dimensions, and long-range electrostatic interactions were treated by the particle mesh Ewald's method (Essmann et al., 1995). The pressure was controlled at 1 atm using Parrinello-Rehman barostat, and the temperature was maintained at 310 K with Langevin's dynamics (Martyna et al., 1994). The short-range and long-range interactions were truncated at 1.2 Å and 1.4 Å, and the LINCS algorithm was used to constrain the bond involving hydrogen atoms (Hess et al., 1997). Equilibration was carried out in NVT, followed by an NPT ensemble with a time step of one fs. The production run with a time step of 2 fs for 50 ns was performed in the NPT ensemble, and snapshots were collected every 100 ps intervals.

Molecular visualization and analyses were performed with the Visual Molecular Dynamics
(VMD) and UCSF Chimera package (Humphrey et al., 1996;Pettersen et al., 2004).  Figure S1. Effect of santalol isomers and tolcapone over TTR WT and TTR V30M stability.     Table S3 and Table S4 for statistical details of lifespan analyses. Figure S4. The relative expression rate of individual subunits of 26S proteasome in TTR WT (left) and TTR V30M (right) worms treated with α-+β-santalol. *p<0.05 vs control.  Data are presented as mean ± SD; ns-not significant, *p<0.05, and **p<0.01 vs. control.