Figure 1.
Representation of a trypsin/SFTI-1 complex and internal hydrogen bonding within SFTI variants during MD.
Ribbon plot of SFTI-1 in complex with trypsin (A) with β-sheets and α-helices coloured in yellow and blue respectively, excluding SFTI-1 which is displayed in magenta. The residues of the catalytic triad of trypsin and the P1 Lys of SFTI-1 are shown in stick models with carbon in green, nitrogen in blue and oxygen in red. The structure of SFTI variants are shown in ball and stick 2D model with intramolecular hydrogen bond networks for (B) SFTI-1, (C) SFTI-FCQR Asp14 and (D) SFTI-FCQR Asn14. Amino acids are labelled with one letter code and residue number in subscript while the frequency of hydrogen bonds per residue is in brackets (rounded to nearest tenth). Carbons, oxygen, nitrogen and sulphur are represented by gray, red, blue and yellow respectively while hydrogens are excluded for clarity. Bond lengths and angles are intentionally unrealistic to enable easy viewing of hydrogen bonds, represented by dotted green line. Only hydrogen bonds occurring in more than 50% of trajectory frames are shown. Data is represented as mean from three independent 5 ns MD trajectories.
Figure 2.
RMSD analysis for SFTI variants during MD.
RMSD values between Cα of SFTI-1, SFTI-FCQR Asp14 and SFTI-FCQR Asn14 during MD and the (A) SFTI-1 starting structure or (B) calculated average simulation structures. (C) Ribbon plot showing the average simulation structures coloured according to Cα RMSD from low to high as blue, purple, magenta, orange, and red, labelled with odd residue numbers. Data is represented as mean from three independent 5 ns MD trajectories.
Table 1.
In silico Internal Hydrogen Bond Analysis of SFTI-FCQR Residue 14 Variants.
Table 2.
Inhibitory Properties of SFTI-1, SFTI-FCQR and SFTI-FCQR Residue 14 Variants.
Figure 3.
Relationship between Ki and number of internal hydrogen bonds.
Plot of the average number of internal (circles), intermolecular (squares) and total (triangles) hydrogen bonds of SFTI-FCQR variants (Asn14, Tyr14, Lys14, Asp14, Gly14, Ala14 and Ser14) from Table 1 versus Morrison Ki values from Table 2.
Figure 4.
Assessment of koff for SFTI-FCQR Asn14.
Lag phases and steady state for inhibitor binding to KLK4: (A) uninhibited reaction progress (B) simultaneous addition of substrate and inhibitor (C) preformed enzyme inhibitor complex. The koff rate was calculated graphically from the absolute difference between the steady states at y = zero. Rates shown are the average of three independent experiments.
Figure 5.
Selective inhibition of serine protease proteolytic activity by SFTI-FCQR Asn14.
Examination of fibrinogen proteolysis by trypsin and kallikreins by SDS-PAGE. Bands were visualised with Coomassie blue staining after resolving on 10% polyacrylamide gels. Images are representative of three separate experiments. Inhibition of KLK4 proteolytic activity by (A) SFTI-FCQR Asp14 and (B) SFTI-FCQR Asn14. Inhibition of trypsin proteolytic activity by (C) SFTI-1 and (D) SFTI-FCQR Asn14. Inhibition of proteolytic activity of (F) KLK12 and (F) KLK14 by SFTI-FCQR Asn14.
Figure 6.
Stability of SFTI variants in contact with prostate cancer cells in vitro.
Residual activity of (A) SFTI-FCQR Asn14 and (B) SFTI-FCQR Lys14 in cell culture media from prostate cancer cells treated with a single dose of inhibitor. Endogenous inhibitors were removed by boiling and centrifugation. Stability was assessed against LNCaP (closed circles), 22Rv1 (triangles), and PC3 cells (open circles). Data are mean ± SEM from three experiments in triplicate.
Figure 7.
Bioavailability of SFTI-FCQR Asn14 in mice.
Serum levels of SFTI-FCQR Asn14 administered at 3 mg/kg via the intravenous (IV), intraperitoneal (IP) routes in mice. Serum half life was 25-28 minutes with 10.0±0.8 nM inhibitor serum levels at 4 hours. The data is expressed as mean ± SEM (IV, n = 3; IP, n = 2).