Development of Clarstatin, a Novel Drug Lead for the Therapy of Autoimmune Uveitis

Abstract

We describe the design, synthesis, and activity of a potent thiourea-bridged backbone cyclic peptidomimetic known as Clarstatin, comprising a 5-amino acid sequence (Q/D)¹-(R/K)²-X³-X⁴-A⁵-(Gln/Asp)¹-(Arg/Lys)²-AA³-AA⁴-Ala⁵-based on a motif called "shared epitope (SE)", specifically present in specific alleles of the HLA-DRB1 gene. This SE binds to a particular site within the proline reach domain (P-domain) of the cell surface-calreticulin (CS-CRT). CS-CRT is a multifunctional endoplasmic reticulum (ER) calcium-binding protein that is located on the cell surface of T cells and triggers innate immune signaling, leading to the development of inflammatory autoimmune diseases. The development of Clarstatin was based on the parent peptide W-G-D¹-K²-S³-G⁴-A⁵- derived from the active region of the SE. Following the design based on the cycloscan method, the synthesis of Clarstatin was performed by the Fmoc solid phase peptide synthesis (SPPS) method, purified by HPLC to 96% homogeneity, and its structure was confirmed by LC-MS. Clarstatin reduced calcium levels in Jurkat lymphocyte cultures, ameliorated uveitis in vivo in the experimental autoimmune uveitis (EAU) mice model, and was safe upon acute toxicity evaluation. These findings identify Clarstatin as a promising lead compound for future drug development as a novel class of therapeutic agents in the therapy of uveitis.

Keywords: backbone cyclization; calreticulin; experimental autoimmune uveoretinitis; inflammatory autoimmune diseases; peptidomimetic; peptomer; thiourea-bridged; uveitis.

Figure 1

Structure of Clarstatin.

Scheme 1

Solid phase synthesis of Clarstatin. Reagents: (a) piperidine/DMF 20%; (b) coupling of Fmoc-[N-(Mtt)δ-aminobutyl]glycine building unit: TBTU/DIEA/DMF; (c) FmocAA + TBTU/DIEA/DMF; (d) coupling of Fmoc-[N-(alloc)δ-aminobutyl]glycine building unit: TBTU/DIEA/DMF; (e) (PPh3)4Pd(0)/N-methylmorpholine/AcOH; (f) isothiocyanate formation: CS₂/HBTU/DIEA/DMF; (g) DCM/TFE/AcOH (7/2/1); (h) DIEA/DMF; (i) TFA/TIS/H2O (95/2.5/2.5).

Figure 2

Analytical HPLC of Clarstatin.

Figure 3

Mass spectrum of Clarstatin.

Figure 4

Ca²⁺ signaling was reduced in Jurkat cells upon Clarstatin treatment in the absence of cytotoxic effects. Changes with time of Ca²⁺ signaling in Jurkat cells in response to (A) LPS (50 µg/mL) and (B) Clarstatin (36 µg/mL); (C) necrotic cell death measured by LDH release in Jurkat cells exposed for 48 h to different Clarstatin concentrations; H₂O₂ treatment was used as a positive cytotoxic control. Values expressed as mean ± SEM (n = 18). p ≤ 0.05 for H₂O₂ vs. control are percent of LDH released in the medium out of the total culture LDH. No significance was found for Clarstatin concentrations compared to control (ANOVA test).

Figure 5

Examination of the therapeutic effect of the cyclic peptide, Clarstatin, in the EAU mice model by histopathology analysis. Representative photographs of the histopathology of retinal sections of C57BL/6J mice 36 days after EAU induction. (A) The wild-type mouse, which was not immunized with IRBP, showed normal retinal structure. (B) The EAU mouse which was immunized with IRBP showed active uveitis with vitreous cells (arrow) and several foci of retinal infiltrates and vasculitis (star). (C) The EAU mouse which was immunized with IRBP and treated with Clarstatin (3.6 μg/kg mice, 50 μL, i.p., twice per week) showed no signs of active uveitis. (D) Quantitation by dose response of the therapeutic effect of Clarstatin based on the EAU score from histopathological sections. EAU scores were assigned on a scale from 0 to 4 according to the extent of inflammation and tissue damage. Each point represents one mouse. The ocular inflammatory response was ameliorated in mice treated with Clarstatin compared to untreated mice. * p < 0.05 *** p < 0.001 (ANOVA test).

Figure 6

Representative histological images of organ slices stained with hematoxylin and eosin in an acute toxicity test of female ICR mice after administration of Clarstatin (10 mg/kg) by i.v. injection for 48 h.