Conformational ligand-directed targeting of calcium-dependent receptors in acute trauma

Abstract

Background: Trauma is a leading cause of mortality, but injury-specific molecular targets remain largely unknown. We hypothesized that distinctive yet unrecognized tissue targets accessible to circulating ligands might emerge during trauma, thereby underscoring a trauma-related proteome.

Methods: We screened a peptide library to discover targets in a porcine model of major trauma: compound femur fracture with hemorrhagic shock. Bioinformatics yielded conserved motifs, and candidate receptors were affinity purified. In silico and in vitro approaches served to investigate possible associations between candidate receptors and calcium, a major component of skeletal muscle and bone. In vivo homing and molecular imaging (PET/MRI and SPECT/CT) studies of the most promising ligand peptide candidate were performed in the porcine model and were also confirmed in a corresponding rat model of major trauma. Optical methodologies and molecular dynamics simulations served to explore the molecular attributes of the ligand-receptor binding.

Findings: Nearly all molecular targets of the selected ligand peptides were calcium-dependent proteins, which become accessible upon trauma. We validated specific binding of homing peptides to these receptors in injured tissues, including CLRGFPALVC:CASQ1, CSEIGVRAC:HSP27, and CRQRPASGC:CALR. Notably, we determined that ligand peptide CRQRPASGC targets an injury-specific calcium-facilitated conformation of calreticulin, enabling specific molecular imaging of trauma.

Conclusions: We conceptually propose the term "traumome" for the functional receptor repertoire that becomes readily amenable for ligand-directed targeting upon major trauma. These preclinical findings pave the way toward clinic-ready targeted theragnostic approaches in the setting of trauma.

Funding: Major funding was provided by the Defense Advanced Research Projects Agency (DARPA).

Keywords: Pre-clinical research; acute trauma; calcium; calreticulin; compound fracture; in vivo screening; peptide library; phage display; receptor; shock; trauma-related proteome.

Figure 1.. Identification and validation of trauma-specific vascular ligands by combinatorial screening.

(A, B) Overview of the in vivo phage display peptide library screening and analysis. (A) Utilization of a porcine model of acute traumatic injury involving compound femur fracture and hemorrhagic shock. Representative images of tissue-section biopsies stained with hematoxylin and eosin demonstrate skeletal muscle damage, hemorrhage, infiltration by inflammatory cells, and fibrin accumulation (scale bar, 250 μm). (B) Systemic administration of an in vivo phage display peptide library (CX₇C and CX₈C) with sample collection from injured and contralateral intact hindlegs at various time points (10, 60, 70, and 120 min, followed by necropsy at 240 min), followed by evaluation of phage particles per sample. (C) Profile of phage clearance after systemic phage library infusion in separate injured pigs (n=3) via arterial blood sample collection at different time points by qPCR per 100 ng of DNA. Data points corresponding to independent animals (wherein two technical replicates were averaged to obtain a single point) are presented as mean ± standard error of the mean (SEM). (D) Phage quantification in the injured and contralateral intact hindlegs at fixed biopsy time points by qPCR per 100 ng of DNA. Data points corresponding to independent animals (wherein two technical replicates were averaged to obtain a single point) are presented as mean + SEM. (E) Phage quantification in hindlegs and control organs after necropsy by qPCR per 100 ng of DNA. Data points corresponding to independent animals (wherein two technical replicates were averaged to obtain a single point) are presented as mean + SEM. (F) Saturation plots of distinct peptide sequences in the biopsy and necropsy samples obtained via random shuffling followed by sampling of the recovered peptide sequences that were detected more than once. Data are presented as the mean of 100 rounds of random shuffling/sampling. (G) Cloning of targeted phage constructs, each displaying a lead peptide candidate (n=23), with their pooling constituting a restricted phage panel. (H) Administration of the restricted phage panel into a separate injured pig (n=1). Phage were quantified in the injured and contralateral intact hindlegs at various biopsy time points by qPCR per 100 ng of DNA. Data points corresponding to technical replicates are presented as mean + SEM and analyzed with two-way ANOVA coupled with post hoc Bonferroni’s multiple comparisons test.

Figure 2.. Trauma-associated ligand-receptors in the context of Ca²⁺ homeostasis.

(A) The traumome—a tentative map of systemically accessible trauma-related proteins, representing the validated candidate receptors (n=18 of 20; DES and COL11A2 not shown) directly (in red) and indirectly (in blue) associated with Ca²⁺. Solid lines indicate a direct physical interaction between a protein and Ca²⁺, and dotted lines indicate a direct physical interaction between two proteins. (B–D) In vitro phage-binding assays with increasing concentrations of cognate peptide for (B) CLRGFPALVC-displaying phage and immobilized recombinant CASQ1, (C) CSEIGVRAC-displaying phage and immobilized recombinant HSP27, and (D) CRQRPASGC-displaying phage and immobilized recombinant CALR. (E) Phage-binding in vitro assays with CLRGFPALVC-displaying phage and immobilized immunocaptured CASQ1 in the absence or presence of EDTA (a Ca²⁺ chelator). (F) Phage-binding in vitro assays with CSEIGVRAC-displaying phage and various immobilized heat-shock proteins. (G) Phage-binding in vitro assays with increasing concentrations of Ca²⁺ with CRQRPASGC-displaying phage and immobilized recombinant CALR. For B–G, phage binding is represented by relative transducing units (TU) as described in Methods. Insertless phage and bovine serum albumin (BSA) served as negative controls for ligand and receptor, respectively. Data points corresponding to technical replicates are presented as mean + SEM and analyzed with either two-way (for E and F) or three-way ANOVA (for B–D and G) coupled with post hoc Bonferroni’s multiple comparisons test. (H) Immunohistochemical (IHC) staining of Ca²⁺ in muscle tissue-section biopsies from injured and non-injured hindlegs of a porcine model of acute trauma 1 and 10 min after injury (scale bar, 250 μm). (I) IHC staining of two direct Ca²⁺-interacting candidate receptors, CASQ1 and CALR, in muscle tissue-section biopsies. Non-injured muscle tissue sections and isotype antibodies were used as negative controls (scale bars, 250 μm).

Figure 3.. Targeting of the CRQRPASGC-CALR ligand-receptor in vivo in the porcine model of major trauma (i.e., femur fracture and/or soft tissue injury) over time.

(A, B) Relative homing of (A) CRQRPASGC-displaying or (B) insertless phage to the fractured hindlegs (n=2 pigs each) following administration, assessed by qPCR per 100 ng of DNA and normalized to the administered sample. Necropsy was performed at 240 min. The contralateral intact hindleg was used as a negative control. Data points corresponding to independent animals (wherein four technical replicates were averaged to obtain a single point) are presented as mean + SEM and analyzed with two-way ANOVA coupled with post hoc Bonferroni’s multiple comparisons test. (C, D) Alanine scanning of CRQRPASGC to assess phage binding of each construct to immobilized recombinant CALR protein in the (C) absence or (D) presence of Ca²⁺. Phage binding is represented by relative TU. BSA and insertless phage were used as negative controls. Data points corresponding to technical replicates are presented as mean + SEM and analyzed with two-way ANOVA coupled with post hoc Bonferroni’s multiple comparisons test. (E) Coronal and axial MRI with regions of interest (ROIs) representing fractured and non-fractured hindlegs of injured pigs for continuous quantification in serial PET/MRI scans. (F) Representative serial axial PET/MRI scans of injured pigs administered with ⁸⁹Zr-labeled CRQRPASGC (n=1 with fracture and n=1 with soft tissue injury only) or ⁸⁹Zr-labeled mutant peptide (CRQRAASGC, red designates the mutation) (n=2 with fracture) at fixed time points. Each individual image is scaled to the same intensity. ⁸⁹Zr-labeled CRQRPASGC in a non-injured pig (n=1) was used as a control. (G) Relative quantification of the ⁸⁹Zr-labeled peptides at the injured ROI represented as percentage of injected dose per gram normalized to the non-injured ROI (contralateral intact hindleg) over six time points (0, 30, 60, 120, 150, 180, 240, 270, and 300 min). Data points corresponding to independent animals are presented as either individual values (orange and black lines) or mean ± SEM (grey line).

Figure 4.. Targeting of the CRQRPASGC-CALR ligand-receptor in vivo in a rat model of acute traumatic injury (i.e., femur fracture and soft tissue injury) over time.

(A) Coronal CT with ROIs representing fractured (right) and non-fractured (left) hindlegs of injured rats for continuous quantification in serial SPECT/CT scans. (B) Planar SPECT scan of both hindlegs in a representative injured rat (n=1) and a non-injured rat (n=1) from 0–18 min following injection with ¹¹¹In-DOTA-labeled CRQRPASGC. (C, D) Quantification of planar SPECT scan from the representative injured rat and non-injured rat. Counts are normalized to the first time point (see Figures S6A and S6B for remaining injured rats). (E) SPECT/CT scans of the injured rat and non-injured rat at fixed time points (60, 90, 180, and 210 min). (F, G) Uptake measured at the (F) injured or (G) non-injured hindleg for all rats (n=3 injured rats and n=1 non-injured rat). Data points corresponding to independent animals are presented as either individual values (black line) or mean ± SEM (orange line).

Figure 5.. Functional and conformational aspects of the interaction between CRQRPASGC and CALR.

(A) In vitro phage-binding assays with CRQRPASGC-displaying phage and immobilized recombinant CALR in the absence or presence of an anti-CALR antibody against its C-terminus (residues A353–E416). Data points corresponding to technical replicates are presented as mean + SEM and analyzed with three-way ANOVA coupled with post hoc Bonferroni’s multiple comparisons test. (B) Steady-state fluorescence emission spectra of CALR in the presence of synthetic CRQRPASGC or mutant peptide (CRQRAASGC) in solution with or without CaCl₂ under excitation at 280 nm (left) and 295 nm (right). (C, D) Circular dichroism (CD) spectrum of CALR in the presence of (C) CRQRPASGC (50 μM) or (D) mutant peptide (50 μM). (E) CD spectra of CALR with high concentration (400 μM) of CRQRPASGC or mutant peptide subtracted by signal attained with low-concentration peptide (50 μM). (F) CD spectra of CALR in CaCl₂ with or without CRQRPASGC (50 μM). Millidegree is abbreviated to mdeg.

Figure 6.. In silico structural analysis of CRQRPASGC binding to CALR and the effects of varying concentrations of Ca²⁺ on the proposed binding site.

(A) Previously determined structural interaction between TAPBP (purple) and CALR (Coulombic surface coloring where red is negative, blue is positive, and white is neutral) as part of the human peptide-loading complex (PDB ID: 6ENY). A close-up view of the CALR-binding region (in the C-terminus) of TAPBP overlayed with the predicted CRQRPASGC structure (cyan) at the shared PASG motif (green) (interaction details at binding interface are unable to be visualized due to absence of amino acid sidechain resolution). (B) Sequence alignment of TAPBP with CRQRPASGC and other similar peptides identified in the injury site from the screenings. Identical (green), conserved (orange), and semi-conserved (yellow) amino acid residues are highlighted. (C) All-atom (non-H) RMSD values and (D) their frequencies for the PASG motif of CRQRPASGC across a 1 μs all-atom explicit-solvent simulation relative to the experimental structure of TAPBP. (E) Overlap of configurations sampled by the PASG motif of CRQRPASGC along the simulation. (F) RMSD value frequencies and (G) per-residue RMSF values of simulated C-terminal amino acid residues (R366–E386) of CALR at different Ca²⁺ concentrations (0, 5, 10, and 20 mM) relative to its experimental structure.