Kvetkina AN et al. (2024), Sea Anemone Kunitz Peptide HCIQ2c1: Structure, ...

XB-ART-61149

Mar Drugs 2024 Dec 02;2212:. doi: 10.3390/md22120542.

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Sea Anemone Kunitz Peptide HCIQ2c1: Structure, Modulation of TRPA1 Channel, and Suppression of Nociceptive Reaction In Vivo.

Kvetkina AN , Oreshkov SD , Mironov PA , Zaigraev MM , Klimovich AA , Deriavko YV , Menshov AS , Kulbatskii DS , Logashina YA , Andreev YA , Chugunov AO , Kirpichnikov MP , Lyukmanova EN , Leychenko EV , Shenkarev ZO .

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TRPA1 is a homotetrameric non-selective calcium-permeable channel. It contributes to chemical and temperature sensitivity, acute pain sensation, and development of inflammation. HCIQ2c1 is a peptide from the sea anemone Heteractis magnifica that inhibits serine proteases. Here, we showed that HCIQ2c1 significantly reduces AITC- and capsaicin-induced pain and inflammation in mice. Electrophysiology recordings in Xenopus oocytes expressing rat TRPA1 channel revealed that HCIQ2c1 binds to open TRPA1 and prevents its transition to closed and inhibitor-insensitive 'hyperactivated' states. NMR study of the 15N-labeled recombinant HCIQ2c1 analog described a classical Kunitz-type structure and revealed two dynamic hot-spots (loops responsible for protease binding and regions near the N- and C-termini) that exhibit simultaneous mobility on two timescales (ps-ns and μs-ms). In modelled HCIQ2c1/TRPA1 complex, the peptide interacts simultaneously with one voltage-sensing-like domain and two pore domain fragments from different channel's subunits, and with lipid molecules. The model explains stabilization of the channel in the open conformation and the restriction of 'hyperactivation', which are probably responsible for the observed analgetic activity. HCIQ2c1 is the third peptide ligand of TRPA1 from sea anemones and the first Kunitz-type ligand of this channel. HCIQ2c1 is a prototype of efficient analgesic and anti-inflammatory drugs.

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Genes referenced: trpa1

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	Figure 1. Analgesic activity of HCIQ2c1 in vivo. (A) The pain threshold in the Hot plate test was detected as latency to withdraw or lick the fore or hind paw. (B) Time-dependent effect of HCIQ2c1 on the volume of paw subcutaneous injected with 0.05% AITC (B1) and Volume Growth Index (%) (B2). (C) Analgesic activity of HCIQ2c1 in a model where pain was induced by subplantar injection of 0.05% AITC. (D) Analgesic activity of HCIQ2c1 in a model where pain was induced by subplantar injection of 6 µg/mouse capsaicin. The pain threshold was detected as: (C1,D1) latency to pain-related response or nociceptive behavior (first licking, tucking, scratching, flicking, or biting the injected hind paw), (C2,D2) time spent tucking the injected paw, (C3,D3) the number of licking the injected paw, and (C4,D4) time spent licking. HCIQ2c1 or saline buffer (control) was administrated intramuscularly 60 min before start of the test (A), or AITC (B,C) or capsaicin (D) injection. Data are presented as mean ± S.E.M. (n = 7). * p < 0.05, p < 0.01, and * p < 0.001 indicate significant differences between the control and HCIQ2c1 groups according to one-way ANOVA/Dunnett’s multiple comparisons test.
	Figure 2. Recombinant HCIQ2c1 affects the diclofenac-evoked currents in X. laevis oocytes expressing rat TRPA1 in the experiment with 30-s HCIQ2c1 preincubation (A,B) and does not affect the currents in the experiment with 90-s pre-pulse of the agonist and second simultaneous 90-s HCIQ2c1+diclofenac pulse (C,D). (A,C) Average current traces normalized to the amplitude of the currents in the time interval labelled “norm.” (n = 12 (A), n = 6–7 (C), different oocytes were recorded, S.E.M. range is shown as the shade around the trace). Three (A) or two (C) consecutive responses (Control, HCIQ2c1, Wash) were measured on each oocyte at 5 min intervals. Direction of the current is shown by the labels “OUT” and “IN”. The application of compounds is shown by bars above the current traces. The amplitude of responses was measured at time points labeled “test”. The concentrations of diclofenac and HCIQ2c1 were 1 mM and 10 µM, respectively. (B,D) The normalized current amplitudes (mean ± S.E.M.). n.s., not significant. * p < 0.05 and ** p < 0.01 indicate significant differences between the “HCIQ2c1” and “Control” data groups with the same direction of currents based on one-sample (B, OUT) and two-sample (B, IN) two-sided t-tests. No significant differences were found for the data presented in panel (D). The non-normalized current traces for the data presented in this figure are shown in Figure S2.
	Figure 3. Recombinant HCIQ2c1 affects the residual currents through rat TRPA1 in X. laevis oocytes after 90-s AITC pulse (A,B) and does not affect the currents in the experiment with 100-s AITC pre-pulse, the second simultaneous 100-s HCIQ2c1+AITC pulse, the third ‘readout’ 100-s AITC pulse, and the final application of the antagonist HC030031 (C,D). (A) Average current traces normalized to the amplitude of the currents in the time interval labelled “norm.” (n = 7–8 (A), n = 6–7 (C), each response was measured on a distinct oocyte, S.E.M. range is shown as the shade around the trace). Direction of the current is shown by the labels “OUT” and “IN”. The application of compounds is shown by bars above the current traces. The amplitude of responses was measured at time points labeled “test” or marked with arrows. The TRPA1 antagonist HC030031 was used as a negative control. The concentrations of AITC, HCIQ2c1, and HC030031 were 100 µM, 10 µM, and 50 µM, respectively. (B,D) The normalized current amplitudes (mean ± S.E.M.). * p < 0.05 and ** p < 0.01 indicate significant differences between the “HCIQ2c1” and “Control” data groups with the same direction of currents based on two-sided t-tests. The only significant difference in panel (D) is the difference in residual current level after application of HC030031. The non-normalized current traces for the data presented in this figure are shown in Figure S3.
	Figure 4. NMR data define the HCIQ2c1 secondary structure. (A) 2D 15N-HSQC spectrum of 0.08 mM 15N-labeled HCIQ2c1 (30 °C, pH 4.5). The resonances of side chain NH2 groups are connected by dashed lines. The system of minor signals is shown in red color. (B–D) The ring-current contributions from the nearby aromatic side chains explain atypical up–field shifts of 1Hδ22 Asn45 and 1HN Gly38 resonances (B), 1H2Cβ and 1H2Cγ resonances of the Lys10 side chain (C), and 1Hβ3 resonance of Cys56 (D). The secondary structure of HCIQ2c1 (E). Elements of the secondary structure were calculated using the STRIDE program [29] from the determined spatial structure of HCIQ2c1 (see below). The β-strands are designated by arrows, α- and 310 helices by rectangles. The L1 and L2 loops are underlined. Possible position of the protease cleavage site is shown by red arrow. The probabilities of the residues to participate in the α-helix or β-strand (Pα and Pβ) were calculated from the chemical shifts in the TALOS-N software [30]. Asterisks indicate the residues with low amplitude of the amide proton temperature gradient (\|Δδ1HN/ΔT\| < 4.5 ppb/°K). Small (<6 Hz), large (>8 Hz), and medium (others) 3JHNHα coupling constants are indicated by empty, filled triangles, and open squares, respectively. Map of NOE contacts (τm = 100 ms) is shown as usual. (F) Topology of the HCIQ2c1 secondary structure. The residues possibly forming a protease binding site are marked.
	Figure 5. The spatial structure and backbone dynamics of HCIQ2c1 in aqueous solution. (A) Two–sided view of the HCIQ2c1 molecule. Positively charged (+His), negatively charged, hydrophobic, and aromatic residues are colored by blue, red, yellow, and green, respectively. The disulfide bonds are shown in orange. (B,C) Two-sided view of the molecular surface of HCIQ2c1. Electrostatic (B) and molecular hydrophobicity [34] (C) potentials are shown. (D) Regions with high-amplitude mobility on the ps–ns time-scale (where S2 < 0.75 or 15N–{1H} NOE < 0.65) are shown in cyan color. (E) Regions with mobility on the μs–ms time-scale are shown in purple (REX ≥ 3.0 s−1 or R1 × R2 > 20.0 s−2) and blue (3 ≥ REX > 0 s−1) colors. The residues demonstrating line-broadening due to intense μs–ms time-scale motions (Cys15 and Gly38) are shown in green color. Regions where signal doubling was observed due to mobility on the millisecond time-scale are shown in red color.
	Figure 6. Best docking solutions of the TRPA1/HCIQ2c1 complex, viewed from the extracellular side. The four subunits of the open rat TRPA1 channel are shown by differently colored surfaces (A–D). Each subunit includes a ¼ of the pore domain (PD; in center) and the voltage-sensing-like domain (VSLD; distal). The HCIQ2c1 backbone is spectrum colored from blue (N-terminus) to red (C-terminus). Disulfide bonds are shown in yellow. The glycans on the VSLDs were modeled in MD but omitted in docking.
	Figure 7. MD snapshot of the best TRPA1/HCIQ2c1 complex (5–1, see Table S5). Colors and designations are the same as in Figure 6. The N-glycan groups attached to Asn749 and Asn755 on the S1–S2 loop of each VSLD are represented as sticks and colored by atom type. (A,B) Top and side view on the simulation system. Membrane lipids are shown as a surface; water and ions are omitted. In (B), the nearby membrane slab is hidden for clarity. (C,D) Close-up top and side views of the TRPA1/HCIQ2c1 complex. Active residues are shown as sticks and colored according to the residue type: positively charged—blue, negatively charged—red, polar—violet, hydrophobic/aromatic—green, cysteines—yellow. Channel residues are italicized and shown in thinner and lighter sticks. POPC lipids and glycine residues are shown with sticks colored by atom type.
	Figure 8. Comparison of HCIQ2c1 with other Kunitz-type peptides. (A) Multiple sequence alignment. Positively and negatively charged residues are indicated by blue and red squares respectively; cysteines are shown in yellow. The green arrow shows a site resistant to proteolytic cleavage, but not all listed peptides demonstrate protease inhibition activity. Cyan boxes indicate the residues involved in TRPV1 inhibition by HCRG21 and APHC1, and regions responsible for the HCIQ2c1 binding to rat TRPA1 in complex 5-1. Magenta boxes indicate the residues involved in interaction with K+-channels. Orange boxes show the residues critical for mambaquaretin-1 (MQ-1) interaction with the type-2 vasopressin receptor. Conserved disulfide bonds and secondary structure elements defining Kunitz-fold are shown. Black arrows and white box indicate the β-strands and α-helix, respectively; wavy lines show the L1 and L2 loops. Sequence identity with HCIQ2c1 (%), PDB codes, and root mean square deviation (RMSD) values calculated over Cα-atoms in regions of conserved secondary structure (19–36, 45–57) are shown on the right. (B) Comparison of the spatial structures of HCIQ2c1 with other Kunitz-type peptides (see legend for color code). The backbones of the peptides are shown as ribbons, cysteines are shown in yellow, and conserved Arg/Lys residues at position P1 of the protease binding site are shown as sticks.