Novel Orthogonally Hydrocarbon-Modified Cell-Penetrating Peptide Nanoparticles Mediate Efficient Delivery of Splice-Switching Antisense Oligonucleotides In Vitro and In Vivo.

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

Pharmacological stimulation of GAL1R but not GAL2R attenuates kainic acid-induced neuronal cell death in the rat hippocampus.

The neuropeptide galanin is widely distributed in the central and peripheral nervous systems and part of a bigger family of bioactive peptides. Galanin exerts its biological activity through three G-protein coupled receptor subtypes, GAL1-3R. Throughout the last 20years, data has accumulated that galanin can have a neuroprotective effect presumably mediated through the activation of GAL1R and GAL2R. In order to test the pharmaceutical potential of galanin receptor subtype selective ligands to inhibit excitotoxic cell death, the GAL1R selective ligand M617 and the GAL2R selective ligand M1145 were compared to the novel GAL1/2R ligand M1154, in their ability to reduce the excitotoxic effects of intracerebroventricular injected kainate acid in rats. The peptide ligands were evaluated in vitro for their binding preference in a competitive (125)I-galanin receptor subtype binding assay, and G-protein signaling was evaluated using both classical signaling and a label-free real-time technique. Even though there was no significant difference in the time course or severity of the kainic acid induced epileptic behavior in vivo, administration of either M617 or M1154 before kainic acid administration significantly attenuated the neuronal cell death in the hippocampus. Our results indicate the potential therapeutic value of agonists selective for GAL1R in the prevention of neuronal cell death.

Rational design of a series of novel amphipathic cell-penetrating peptides.

A series of novel, amphipathic cell-penetrating peptides was developed based on a combination of the model amphipathic peptide sequence and modifications based on the strategies developed for PepFect and NickFect peptides. The aim was to study the role of amphipathicity for peptide uptake and to investigate if the modifications developed for PepFect peptides could be used to improve the uptake of another class of cell-penetrating peptides. The peptides were synthesized by solid phase peptide synthesis and characterized by circular dichroism spectroscopy. Non-covalent peptide-plasmid complexes were formed by co-incubation of the peptides and plasmids in water solution. The complexes were characterized by dynamic light scattering and cellular uptake of the complexes was studied in a luciferase-based plasmid transfection assay. A quantitative structure-activity relationship (QSAR) model of cellular uptake was developed using descriptors including hydrogen bonding, peptide charge and positions of nitrogen atoms. The peptides were found to be non-toxic and could efficiently transfect cells with plasmid DNA. Cellular uptake data was correlated to QSAR predictions and the predicted biological effects obtained from the model correlated well with experimental data. The QSAR model could improve the understanding of structural requirements for cell penetration, or could potentially be used to predict more efficient cell-penetrating peptides.

Identification of cell-penetrating peptides that are bactericidal to Neisseria meningitidis and prevent inflammatory responses upon infection.

Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

Cell-penetrating peptides-based strategies for the delivery of splice redirecting antisense oligonucleotides.

Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs. Cell-penetrating peptides (CPPs) now appear as promising non-viral delivery vector for non-permeant biomolecules. We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery.

Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides.

Conjugates of cell-penetrating peptides (CPP) and splice redirecting oligonucleotides (ON) display clinical potential as attested by in vivo experimentation in murine models of Duchenne muscular dystrophy. However, micromolar concentrations of these conjugates are required to obtain biologically relevant responses as a consequence of extensive endosomal sequestration following endocytosis. Recent work from our group has demonstrated that appending stearic acid to CPPs increases their efficiency and that the inclusion of pH titrable entities leads to further improvement. Moreover, these modified CPPs form non covalent complexes with charged ON analogs or siRNAs, which allows decreasing the concentrations of ONs by nearly one log. These modified CPPs and the parent peptides have been compared here in the same in vitro model in terms of cell uptake, trafficking and splicing redirection activity. The increased splicing redirection activity of our modified CPPs cannot be explained by differences in cell uptake but rather by their enhanced ability to escape from endocytic vesicles. Accordingly, a clear correlation between membrane destabilizing activity and splicing redirection was observed using a liposome leakage assay. Studies of cellular trafficking for the most active PF6:ON complexes indicate uptake by clathrin-mediated endocytosis using either FACS cell uptake or a splicing redirection functional assay. Acidification of intracellular vesicles and membrane potential were found important for splicing redirection but not for cell uptake. These results do confirm that the increased potency of PF6:ON complexes is not due to the use of a non endocytic route of cell internalization as proposed for some CPPs.

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation.

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo.

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

The native copper- and zinc-binding protein metallothionein blocks copper-mediated Abeta aggregation and toxicity in rat cortical neurons.

BACKGROUND: A major pathological hallmark of AD is the deposition of insoluble extracellular beta-amyloid (Abeta) plaques. There are compelling data suggesting that Abeta aggregation is catalysed by reaction with the metals zinc and copper. METHODOLOGY/PRINCIPAL FINDINGS: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Abeta1-40 and Abeta1-42. This action of MT-2A appears to involve a metal-swap between Zn7MT-2A and Cu(II)-Abeta, since neither Cu10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Abeta aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Abeta induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons. CONCLUSIONS/SIGNIFICANCE: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Abeta aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Abeta leaving a metal-free Abeta that can readily bind metals again, we believe that MT-2A might represent a different therapeutic approach as the metal exchange between MT and Abeta leaves the Abeta in a Zn-bound, relatively inert form.

Proteins in the insulin-secreting cell line MIN6 bind the imidazoline compound BL11282.

The imidazoline BL11282 stimulates insulin release and alters islet proteomes. Subcellular fractions of MIN6 cells showed that the membrane fraction exhibited binding to BL11282 on a Biacore chip and to BL11282-labelled magnetic beads. Bound material extracted from the beads showed a approximately 50 kDa differential band upon SDS-PAGE and a weaker 100 kDa band. The former was sensitive to competitive removal by preincubation of the fraction with BL11282, then highlighting the approximately 100 kDa band. Masspectrometric analysis revealed the approximately 50 kDa band to be EF1A and the approximately 100 kDa band to be glucose regulated P94, both of interest in insulin synthesis and secretion.

Oxidative switches in functioning of mammalian copper chaperone Cox17.

Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox17(0S-S) binds co-operatively to four Cu+; Cox17(2S-S), with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox17(3S-S), with three disulfide bridges, does not bind to any metal ions. The E(m) (midpoint redox potential) values for two redox couples of Cox17, Cox17(3S-S)<-->Cox17(2S-S) (E(m1)) and Cox17(2S-S)<-->Cox17(0S-S) (E(m2)), were determined to be -197 mV and -340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox17(0S-S) and Cox17(2S-S), which is slightly shifted towards Cox17(0S-S). In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox17(2S-S), enabling retention of Cox17(2S-S) in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox17(2S-S), capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper-thiolate cluster, which may provide safe storage of an excess of copper ions.

The possible mechanisms by which phanoside stimulates insulin secretion from rat islets.

We recently showed that phanoside, a gypenoside isolated from the plant Gynostemma pentaphyllum, stimulates insulin secretion from rat pancreatic islets. To study the mechanisms by which phanoside stimulates insulin secretion. Isolated pancreatic islets of normal Wistar (W) rats and spontaneously diabetic Goto-Kakizaki (GK) rats were batch incubated or perifused. At both 3 x 3 and 16 x 7 mM glucose, phanoside stimulated insulin secretion several fold in both W and diabetic GK rat islets. In perifusion of W islets, phanoside (75 and 150 microM) dose dependently increased insulin secretion that returned to basal levels when phanoside was omitted. When W rat islets were incubated at 3 x 3 mM glucose with 150 muM phanoside and 0 x 25 mM diazoxide to keep K-ATP channels open, insulin secretion was similar to that in islets incubated in 150 microM phanoside alone. At 16 x 7 mM glucose, phanoside-stimulated insulin secretion was reduced in the presence of 0 x 25 mM diazoxide (P<0 x 01). In W islets depolarized by 50 mM KCl and with diazoxide, phanoside stimulated insulin release twofold at 3 x 3 mM glucose but did not further increase the release at 16 x 7 mM glucose. When using nimodipine to block L-type Ca2+ channels in B-cells, phanoside-induced insulin secretion was unaffected at 3 x 3 mM glucose but decreased at 16 x 7 mM glucose (P<0 x 01). Pretreatment of islets with pertussis toxin to inhibit exocytotic Ge-protein did not affect insulin response to 150 microM phanoside. Phanoside stimulated insulin secretion from Wand GK rat islets. This effect seems to be exerted distal to K-ATP channels and L-type Ca2+ channels, which is on the exocytotic machinery of the B-cells.

Cox17, a copper chaperone for cytochrome c oxidase: expression, purification, and formation of mixed disulphide adducts with thiol reagents.

Copper chaperone for cytochrome c oxidase (Cox17) is a 7 kDa copper-binding protein, which facilitates incorporation of copper ions into Cu(A) site of cytochrome c oxidase. Cox17 contains six conserved Cys residues and occurs in three different oxidative states, which display different metal-binding properties and stability. In the present study, we have elaborated technologies for production of partially oxidized human recombinant Cox17 in a bacterial expression system and purification of fully oxidized Cox17. For this purpose we used Escherichia coli Origami strain, which is deficient in thioredoxin and thioredoxin reductase systems and allows formation of disulfide bonds in cytoplasmic proteins. Fully oxidized Cox17 was purified by a simplified two-step procedure including gel filtration and cation exchange chromatography. By using mass spectrometry we demonstrated that application of 2-mercaptoethanol (2-ME) during purification leads to formation of its mixed disulfide adducts with Cox17. Moreover, partially reduced Cox17 can form mixed disulfide adducts also with the cellular reducing agent glutathione, which abolishes copper-binding ability of partially reduced Cox17.

Human Sco1 functional studies and pathological implications of the P174L mutant.

The pathogenic mutant (P174L) of human Sco1 produces respiratory chain deficiency associated with cytochrome c oxidase (CcO) assembly defects. The solution structure of the mutant in its Cu(I) form shows that Leu-174 prevents the formation of a well packed hydrophobic region around the metal-binding site and causes a reduction of the affinity of copper(I) for the protein. K(D) values for Cu(I)WT-HSco1 and Cu(I)P174L-HSco1 are approximately 10(-17) and approximately 10(-13), respectively. The reduction potentials of the two apo proteins are similar, but slower reduction/oxidation rates are found for the mutant with respect to the WT. The mitochondrial metallochaperone in the partially oxidized Cu(1)(I)Cox17(2S-S) form, at variance with the fully reduced Cu(4)(I)Cox17, interacts transiently with both WT-HSco1 and the mutant, forming the Cox17/Cu(I)/HSco1 complex, but copper is efficiently transferred only in the case of WT protein. Cu(1)(I)Cox17(2S-S) indeed has an affinity for copper(I) (K(D) approximately 10(-15)) higher than that of the P174L-HSco1 mutant but lower than that of WT-HSco1. We propose that HSco1 mutation, altering the structure around the metal-binding site, affects both copper(I) binding and redox properties of the protein, thus impairing the efficiency of copper transfer to CcO. The pathogenic mutation therefore could (i) lessen the Sco1 affinity for copper(I) and hence copper supply for CcO or (ii) decrease the efficiency of reduction of CcO thiols involved in copper binding, or both effects could be produced by the mutation.

Lysophosphatidic acid binds to and activates GPR92, a G protein-coupled receptor highly expressed in gastrointestinal lymphocytes.

Here, the ligand binding, activation, and tissue distribution of the orphan G protein-coupled receptor (GPCR) GPR92 were studied. GPR92 binds and is activated by compounds based on the lysophosphatidic acid (LPA) backbone. The binding of LPA to GPR92 was of high affinity (K(D) = 6.4 +/- 0.9 nM) and led to an increase in both phosphoinositide hydrolysis and cAMP production. GPR92 is atypical in that it has a low sequence homology with the classic LPA(1-3) receptors (21-22%). Expression of GPR92 is mainly found in heart, placenta, spleen, brain, lung, and gut. Notably, GPR92 is highly expressed in the lymphocyte compartment of the gastrointestinal tract. It is the most abundant GPCR activated by LPA found in the small intestinal intraepithelial CD8+ cytotoxic T cells.

Addition of arylboronic acids to symmetrical and unsymmetrical azo compounds.

[reaction: see text] The addition of aryl- and heteroarylboronic acids to azo compounds is described. Copper salt catalysis was necessary to perform the reaction under mild conditions and high yields. Excellent regioselectivity was observed in addition to unsymmetrical azo compounds.

A novel form of neurotensin post-translationally modified by arginylation.

A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the gamma-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide "arginylated neurotensin" (R-NT, N-(neurotensin-C5-4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1.

Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity.

Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2 approximately four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form alpha-helical secondary structure in TFE-water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% alpha-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.

Pharmacological characterization of relaxin-3/INSL7 receptors GPCR135 and GPCR142 from different mammalian species.

Relaxin-3 has recently been identified as a ligand for two structurally related G-protein-coupled receptors, human GPCR135 and GPCR142. This current study reports the characterization of mouse and rat GPCR135 as well as GPCR142 from mouse, monkey, cow, and pig at the molecular and pharmacological levels. Mouse and rat GPCR135 exhibit high homology (>85%) to the human GPCR135 and have very similar pharmacological properties to that of the human GPCR135. Human and mouse/rat relaxin-3 both bind to and activate mouse, rat, and human GPCR135 at high affinity with IC(50) or EC(50) values close to 0.5 nM. In contrast, the mouse GPCR142 is less well conserved (74% homology) with human GPCR142. The rat GPCR142 gene was found to be a pseudogene. We further cloned GPCR142 genes from monkey, cow, and pig and found that they are highly homologous (>84%) to human GPCR142. Pharmacological characterization of GPCR142 from different species demonstrated that relaxin-3 binds to GPCR142 from different species at high affinity (IC(50) < 5 nM). However, relaxin-3 does not stimulate a Ca(2+) response in cells coexpressing Galpha(16) and mouse GPCR142, whereas it does for cells expressing GPCR142 from other species tested. Our results suggest that GPCR142 may have a diminished role as a receptor for relaxin-3 in rodents, or perhaps GPCR142 functions as a receptor for another ligand in nonrodents. Boels and Schaller recently reported bradykinin as a ligand for GPCR142 (also known as GPR100). In this report, we demonstrate that bradykinin activates neither GPCR135 nor GPCR142, whereas relaxin-3 does.

INSL5 is a high affinity specific agonist for GPCR142 (GPR100).

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of (125)I-relaxin-3 to GPCR142 with a high affinity (K(i) = 1.5 nM). In a saturation binding assay, (125)I-INSL5 binds GPCR142 with a K(d) value of 2.5 nM. In functional guanosine (gamma-thio)-triphosphate binding and cAMP accumulation assays, INSL5 potently activates GPCR142 with EC(50) values of 1.3 and 1.2 nM, respectively. In addition, INSL5 stimulates Ca(2+) mobilization in HEK293 cells expressing GPCR142 and G alpha(16). Overall, INSL5 behaves as an agonist for GPCR142 similar to relaxin-3. However, unlike relaxin-3, which is also a potent agonist for GPCR135 and LGR7, INSL5 does not activate either GPCR135 or LGR7. INSL5 inhibits (125)I-relaxin-3 binding to GPCR135 with a low potency (K(i) = 500 nM). A functional assay shows that INSL5 (1 microm) is a weak antagonist for GPCR135. In addition, INSL5 (up to 1 microm) shows no affinity or activity at LGR7 or LGR8 either in a binding assay or a bio-functional assay. Previously, we have demonstrated that GPCR142 mRNA is expressed in peripheral tissues, particularly in the colon. Here we show that INSL5 mRNA is expressed in many peripheral tissues, similar to GPCR142. The high affinity interaction between INSL5 and GPCR142 coupled with their co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for GPCR142.