Like other canonical inhibitors, SFTI-1 interacts with a cognate enzyme (i.e. with trypsin) <i>via</i> its solvent-exposed binding loop between Cys³ and Cys¹¹.

<p>The central peptide bond between Lys⁵ (P₁) and Ser⁶ (P₁′) is known as a reactive site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Luckett1" target="_blank">[17]</a>. The adjacent residues placed on the left side of this bond are marked with non-prime P (P₂, P₃, … P_n etc), whereas, on the right side, with prime P (P₂′, P₃′, … P_n etc) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Schechter1" target="_blank">[18]</a>. Corresponding enzyme substrate binding pockets are called non-prime (S₁, S₂, S₃, …, S_n) and prime (S₁′, S₂′, S₃′, …,S_n′) sites, respectively.</p

Inhibitory activities of monocyclic SFTI-1(<b>I</b>) and its analogues at pH 8.1 and 37°C.

<p>*- the K_i values were calculated using the online IC₅₀-to-<i>K_i</i> converter tool assuming competitive inhibition (BotDB Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089465#pone.0089465-Shibatani1" target="_blank">[22]</a>); N/D – not determined (very low activity). The highest activities (the lowest IC₅₀ values) are in bold. Z-LLL-CHO was used as the positive control (mean±S.E.M., n = 3).</p

Inhibition of T-L activity of latent human 20S proteasome by Z-LLL-CHO, monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 52 µm).

<p>Inhibition of T-L activity of latent human 20S proteasome by Z-LLL-CHO, monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 52 µm).</p

The rate of substrate Suc-LLVY-AMC hydrolysis (a release of AMC molecule) with control and inhibitor V- treated human 20S versus the substrate concentration (A), the enzyme concentration was kept constant at 2.7 nm; the double reciprocal Lineweaver-Burk plot (B).

<p>The rate of substrate Suc-LLVY-AMC hydrolysis (a release of AMC molecule) with control and inhibitor V- treated human 20S versus the substrate concentration (A), the enzyme concentration was kept constant at 2.7 nm; the double reciprocal Lineweaver-Burk plot (B).</p

Inhibition of human 20S C-L activity by Z-LLL-CHO, BPTI monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 80 µm).

<p>Inhibition of human 20S C-L activity by Z-LLL-CHO, BPTI monocyclic SFTI-1 and its analogues ([E] = 2.7 nm; [S] = 80 µm).</p

Inhibition of human 20S ChT-L activity by the selected peptides, [E] = 4.4 nm (0.5 µg/200 µL); [S] = 52.4 µm.

<p>Inhibition of human 20S ChT-L activity by the selected peptides, [E] = 4.4 nm (0.5 µg/200 µL); [S] = 52.4 µm.</p

Mass spectra of analogue III (M.W = 1590.9 Da; Cm = 103.0 µm) and IX (M.W = 1463.7 Da; Cm = 134.0 µm) recorded before incubation and after 4 hours, 24 hours and 48 hours incubation with SDS-activated yeast 20S proteasome ([E] = 0.18 µm).

<p>Mass spectra of analogue III (M.W = 1590.9 Da; Cm = 103.0 µm) and IX (M.W = 1463.7 Da; Cm = 134.0 µm) recorded before incubation and after 4 hours, 24 hours and 48 hours incubation with SDS-activated yeast 20S proteasome ([E] = 0.18 µm).</p

Assays in 50m Tris-HCl buffer without SDS (A) analogue V; (B) analogue IX.

<p>Both peptides stimulate ChT-L activity of latent yeast 20S proteasome; [E] = 0.9 nm; [Suc-LLVY-AMC] = 5.2 µm; incubation time 30 min. at 37°C. Data for analogue III are not presented.</p

Inhibition of chymotrypsin-like (ChT-L) and caspase-like (C–L) activities of SDS-activated human 20S ([E] = 2.7 nm) by BPTI and Z-LLL-CHO (A).

<p>Inhibition of trypsin-like (T-L) activity of latent human 20S ([E] = 2.7 nm) (<b>B</b>). Incubation time was about 30 min. at 37°C.</p