Sustained Activation of CLR/RAMP Receptors by Gel-Forming Agonists

Abstract

Background: Adrenomedullin (ADM), adrenomedullin 2 (ADM2), and CGRP family peptides are important regulators of vascular vasotone and integrity, neurotransmission, and fetoplacental development. These peptides signal through CLR/RAMP1, 2, and 3 receptors, and protect against endothelial dysfunction in disease models. As such, CLR/RAMP receptor agonists are considered important therapeutic candidates for various diseases. Methods and Results: Based on the screening of a series of palmitoylated chimeric ADM/ADM2 analogs, we demonstrated a combination of lipidation and accommodating motifs at the hinge region of select peptides is important for gaining an enhanced receptor-activation activity and improved stimulatory effects on the proliferation and survival of human lymphatic endothelial cells when compared to wild-type peptides. In addition, by serendipity, we found that select palmitoylated analogs self-assemble to form liquid gels, and subcutaneous administration of an analog gel led to the sustained presence of the peptide in the circulation for >2 days. Consistently, subcutaneous injection of the analog gel significantly reduced the blood pressure in SHR rats and increased vasodilation in the hindlimbs of adult rats for days. Conclusions: Together, these data suggest gel-forming adrenomedullin analogs may represent promising candidates for the treatment of various life-threatening endothelial dysfunction-associated diseases such as treatment-resistant hypertension and preeclampsia, which are in urgent need of an effective drug.

Keywords: CLR/RAMP receptor; RAMP2; adrenomedullin; endothelium; liquid gel; treatment-resistant hypertension; vasodilation.

Figure 1

Alignment of ADM family peptides and chimeric analogs. The alignment includes wild-type peptides and Analogs A-1 to A9 and Analogs B-1 to B-6. In chimeric analog sequences, residues derived from CGRP, ADM, and ADM2 are indicated by blue, red, and black letters, respectively. The disulfide ring-forming cysteines are indicated by a yellow background. The N-terminal modifications, including lysine-conjugated palmitoylation (Pal-K or K(pal)) and mini-PEGylation (abbreviated as mPEG), are indicated by brown letters. Dash lines are added in select sequences to improve alignment. Acetylation is abbreviated as Ace. The putative activation, binding, and junctional regions of these peptides are indicated by blue, green, and red horizontal bars above the alignment, respectively.

Figure 2

Alignment of analogs with an N-terminal sequence modification or a truncation at the junctional region. The alignment includes CGRP (blue letters), adrenomedullin (red letters), and adrenomedullin 2 (black letters) as well as Analogs C-1 to C-9 and Analogs D-1 to D-8. Cysteine residues that form a disulfide ring are indicated by a yellow background. A putative collagen-binding motif is indicated by purple letters. The origin of individual residues in chimeric analogs is indicated by text colors. The N-terminal modifications, including lysine-conjugated palmitoylation (Pal-K or K(pal)) and mini-PEGylation (abbreviated as mPEG), are indicated by brown letters. Sequence gaps are indicated by dash lines. The putative activation, binding, and junctional regions of these analogs are indicated by blue, green, and red horizontal bars above the alignment, respectively. The motif corresponding to the hinge region of CGRP is indicated by a blue rectangle box.

Figure 3

Analogs with enhanced receptor-activation activity potently stimulate the proliferation and survival of primary HLME cells. Analogs A-5, B-1, B-3, C-3, C-4, C-5, and C-7 significantly increased (A) the proliferation of primary human lymphatic microvascular endothelial (HLME) cells in a medium with 2% FBS and (B) the survival of HLME cells in medium with 0.1% FBS. HLME cells were cultured for 72 h, and cell viability was quantified with the MTT assay. The wild-type peptides (ADM1-52, ADM14-52, and ADM2) and a nonlipidated analog (Analog B-6) had negligible effects on HLME cell proliferation and survival. Analogs with an enhanced CLR/RAMP2-activation activity are indicated by red letters. Data are mean ± SEM of quintuplicate samples. Similar results were obtained in at least three different experiments. *, Significantly different from controls.

Figure 4

Analog C-6 self-assembles to form gels in situ, and the analog solution has a high viscosity. (A,B) Representative pictures of 11% Analog C-6 gel solution at 30 min after dissolution. The gel mass is indicated by blue arrows. (C) The relative viscosity of a 5% glucose solution (water solution), 1% Tween 20 solution, 0.5% and 5% wild-type ADM solutions in 5% glucose, and 0.5% and 5% Analog C-6 solutions in 5% glucose. The relative viscosity was measured by a Rheosense viscometer *, Significantly different from the water control. #, Significantly different from the wild-type ADM solution with the same peptide level (i.e., 0.5% or 5%).

Figure 5

Gel formation hinders the passage of Analog C-6 molecules through Centricon membrane filters. To determine whether gel formation reduces the peptide’s freedom to move, we analyzed the passage of (A) CGRP, (B) ADM, and (C) Analog C-6 molecules through the Centricon membrane filter (MW cutoff: 30,000). The testing solutions included 0.1% peptide in 5% glucose (0.1% solution), 0.1% peptide in saline solution as well as 5% and 20% peptide in 5% glucose (5% and 20% solution). For 5% and 20% solution, one-milligram aliquots of the peptides were first dissolved at the target concentration and diluted to the 0.1% level 10 min later. Aliquots of these solutions were then dispensed into individual Centricon columns before centrifugation for 15 min (2000× g). Levels of peptides in the elute were determined by specific CGRP or ADM EIA. *, Significantly different from the 0.1% solution control group. Data are mean ± SEM of triplicate samples.

Figure 6

Subcutaneous administration of Analog C-6 gel solution leads to the sustained presence of the peptide in the circulation of rats. The circulating level of Analog C-6 was significantly increased in the circulation from 8 to 48 h after injection. An aliquot of Analog C-6 gel solution (32 mg in 16% solution) was delivered subcutaneously in male adult Sprague–Dawley rats, and blood samples were collected at pre-dose, 8, 24, 48, 96, and 192 h after injection. The peptide level was determined by specific ADM EIA. *, Significantly different from controls at 0 h. Data are mean ± SEM of three separate animals.

Figure 7

The Analog C-6 gel solution has a prolonged effect on blood pressure in SHR rats. Subcutaneous injection of Analog C-6 gel solution led to the significant reduction of (A) systolic blood pressure at 2, 6, 24, and 48 h after administration and (B) diastolic blood pressure at 2, 6, and 24 h after administration in male SHR rats (8%; 8 mg/100 µL injection). On the other hand, Analog C-6 gel injection only affected (C) the heart rate 48 h after injection. The hemodynamics was recorded on 0, 2, 6, 24, and 48 h after drug administration. Data are mean ± SEM of five separate animals. In addition, the change of hemodynamics of a representative control animal, which received saline injection, is included for comparison (red lines) *, Significantly different from the measurement at 0 h.

Figure 8

Analog C-6 gel solution has a prolonged effect on dermal blood flow in the hindlimbs of Sprague–Dawley rats. (A) Percentage changes of dermal blood flow in the left and right hindlimbs at 0, 1, 2, 4, 6, 24, 48, and 72 h after a subcutaneous injection of gel solution (8 mg in 8% solution) in the left hindlimb of anesthetized adult male Sprague–Dawley rats. The rats received the injection after the basal scans with a Doppler imager at the beginning of the experiment. The blood flow in the hindlimbs was again scanned at 1, 2, 4, 6, 24, 48, and 72 h after the start of the experiment. Data are reported as percent change from the average of baseline scans and are mean ± SEM of six separate animals. *, Significantly different from the basal level at 0 h. (B) The difference of changes in blood flow between the left (treatment side) and right (control side) hindlimbs at 0, 1, 2, 4, 6, 24, 48, and 72 h after the injection of gel solution at the left hindlimb. *, significantly different between the two hindlimbs. (C) Representative scans of dermal blood flow at different time points in animals that received an Analog C-6 gel solution injection.

Figure 9

Illustration of the putative position of the hinge region in CGRP/ADM family peptides and analogs. The interaction of ADM analogs and CLR/RAMP receptors could be analogous to that between CGRP and CLR/RAMP1 (RCSB protein data bank [PDB] structure 6E3Y). The structure view of CGRP-CLR/RAMP1 complex 6E3Y is depicted from two different angles, and it includes CGRP (red), RAMP1 (pink), and CLR (yellow) components. The unresolved structure at the hinge region is indicated by a white circle. Residues Val23 and Phe27, which are neighboring residues of the hinge region (Lys24-Asn26), and the C-terminal Phe37 of CGRP are indicated by arrows.