IMMODULATOR PEPTIDES COVALENTLY MODIFIED WITH SMALL MOLECULES

Abstract

This invention provides modified IGFBP-derived peptides—collectively termed “immodulator peptides”—and related compositions and methods. Chemical modifications to peptides using small molecules, and sequence extensions to immodulator core sequences exhibit new and surprising biological activities. The invention builds the combinatorial power of the immodulator peptide class further by demonstrating which core sequences bind metal or glycosaminoglycans such as heparin. The invention discloses some surprising biological properties of compositions derived from these modifications, including a host of new therapeutic and diagnostic utilities (e.g. immune modulation of TLR signaling, enhanced collagen synthesis by skin fibroblasts, and synergy with a RIG-1 agonist in killing melanoma cells). The invention also teaches methods for enhancing previously disclosed uses of immodulator peptides by showing how the modifications of the invention can boost the efficacy of immodulator peptides in a model of burn trauma.

Claims

1. A modified peptide, 20-60 amino acids in length, comprising: (i) an insulin-like-growth-factor-binding-protein-derived immodulator core amino acid sequence comprising an amino acid sequence corresponding to SEQ ID NO:1 or SEQ ID NO:2; and (ii) a small molecule of molecular mass less than one thousand daltons linked covalently to the amino terminus of the amino acid sequence. wherein said small molecule is a carboxylic acid that is not an amino acid or biotin.

2. The modified peptide according to claim 1, wherein said terminally-linked carboxylic acid is selected from the group consisting of: oleic acid, eicosapentanoic acid, lauric acid, decanoic acid, lignoceric acid, docosahexanoic acid, 2-hydroxy-2-decenoic acid, phenolic acids, anthraquinones, pentacyclic triterpenoids, retinoic acids, adapalene, bexarotene, rhein, proprionic acids, keto acids, cinnamic acids, aromatic carboxylic acids, indoleacetic acids, xanthenes, xanthones, fenofibric acid, valproic acid, 2-hexyl-4-pentynoic acid, 2,7-dichlorodihydro-fluorescein diacetate, indolyl-carboxylic acids, ibuprofen, artemisinic acid, metallocene carboxylic acids, and bromopyruvic acid.

3. The modified peptide according to claim 1 or claim 2, wherein the amino acid sequence of said immodulator core amino acid sequence consists of the amino acid sequence of any of SEQ ID NOs:3-8.

4. The modified peptide according to any one of claims 1 to 3, wherein the amino acid sequence of said immodulator core amino acid sequence is carboxyterminally extended by the amino acid sequence ZV, wherein Z is any amino acid except L-cysteine or L-alanine.

5. The modified peptide according to claim 1, further comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:10-22.

6. The modified peptide according to any one of claims 1 to 5, wherein the modified peptide is complexed with a metal selected from the group consisting of: ferrous iron, ferric iron, ferrocene, ferrocene derivative, zinc, copper, vanadium, ruthenium, cobalt, titanium, manganese, and calcium.

7. The modified peptide according to any one of claims 1 to 6, wherein the modified peptide is complexed with a glycosaminogycan selected from the group consisting of: heparin, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, and hyaluronate.

8. A pharmaceutical composition comprising a therapeutically effective amount of the modified peptide according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. A method for treating a subject suffering from immune dysfunction or perturbation, said method comprising administering to said subject a therapeutically effective dose of the modified peptide according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 8, wherein said therapeutically effective dose of the modified peptide is from about 0.01 mg/kg/day to about 50 mg/kg/day.

10. The method according to claim 9, wherein the administration is via an intramuscular, subcutaneous, parenteral, transdermal, intranasal, intravenous, or intrathecal route.

11. The method according to claim 9, further comprising co-administration of a helper molecule that modulates the activity of cellular antiviral defenses, said helper selected from the group consisting of modulators of RIG-I, MDA5, MAVS, STING, IRF3, STAT1, STAT3, TBK1, PACT, LGP2, NFkappaB, DNA methylases and toll-like receptors.

12. Use of the modified peptide according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 8 in the treatment of a subject suffering from immune dysfunction or perturbation.

13. Use of the modified peptide according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 8 in the manufacture of a medicament for treatment of a subject suffering from immune dysfunction or perturbation.

14. Use of the modified peptide according to claim 12, further comprising co-administration of a helper molecule that modulates the activity of cellular antiviral defenses, said helper selected from the group consisting of modulators of RIG-I, MDA5, MAVS, STING, IRF3, STAT1, STAT3, TBK1, PACT, LGP2, NFkappaB, DNA methylases and toll-like receptors.

15. An in vitro method for measuring the potency of the modified peptide according to any one of claims 1 to 7, the method comprising determining the ability of the modified peptide or modified peptide conjugate to modulate Toll-like receptor (TLR) signaling, retinoic acid-inducible gene I (RIG-I) signaling, apoptosis, or collagen synthesis in cultured cells.

Description

DETAILED DESCRIPTION

[0025] The terms “subject” and “individual”, as used herein, refer to a vertebrate individual, including avian and mammalian individuals, and more particularly to sport animals (e.g., dogs, cats, and the like), agricultural animals (e.g., cows, horses, sheep, and the like), and primates (e.g., humans).

[0026] The term “treatment” is used herein as equivalent to the term “alleviating”, which, as used herein, refers to an improvement, lessening, stabilization, or diminution of a symptom of a disease or immune perturbation. “Alleviating” also includes slowing or halting progression of a symptom.

[0027] As used herein, “co-administration”, “in conjunction with”, “concurrent”, or “concurrently”, as used interchangeably herein, refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during or after delivery of the other treatment modality to the subject.

[0028] Proteinogenic amino acids are amino acids that are incorporated biosynthetically into proteins during the process of translation by ribosomes inside a living cell. The proteinogenic amino acids are: alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan and tyrosine.

[0029] This invention claims a substantially purified 20-60 amino acid synthetic peptide comprising an insulin-like-growth-factor-binding-protein-derived immodulator core sequence. For the purposes of this invention, the “core sequence” comprised in the claimed immodulator peptides is either SEQ ID NO:1 or SEQ ID NO:2. This core sequence represents conserved residues derived from IGFBPs 1 through 6, and serves as the scaffold for immodulator peptides. In some embodiments, the above core sequence is C-terminally extended by the dipeptide ZV, where Z is any amino acid except cysteine. More preferably, Z is any amino acid except cysteine or L-alanine.

[0030] This invention provides a peptide having an amino terminus formed by covalent linkage to a carboxylic acid of molecular mass less than one thousand daltons, preferably less than five hundred daltons, wherein said carboxylic acid that is not an amino acid or biotin, and is selected from a group consisting of oleic acid, eicosapentanoic acid, lauric acid, decanoic acid, lignoceric acid, docosahexanoic acid, 2-hydroxy-2-decenoic acid, phenolic acids, anthraquinones, pentacyclic triterpenoids, retinoic acids, adapalene, bexarotene, rhein, proprionic acids, keto acids, cinnamic acids, aromatic carboxylic acids, indoleacetic acids, xanthenes, xanthones, fenofibric acid, valproic acid, 2-hexyl-4-pentynoic acid, 2,7-dichlorodihydro-fluorescein diacetate, indolyl-carboxylic acids, ibuprofen, artemisinic acid metallocenes such as ferrocene carboxylic acid and bromopyruvic acid.

[0031] The invention provides a modified immodulator peptide according to the above, wherein said immodulator core sequence is selected from a group consisting of SEQ ID NOs: 3-8. In some embodiments, said core sequence is C-terminally extended by the dipeptide ZV wherein Z is any amino acid except cysteine. Preferably Z is glycine, D-alanine, or any of the D- or L- isomers of serine, arginine, lysine, aspartic acid, glutamic acid or phenylalanine.

[0032] The invention further claims an immodulator peptide according to the descriptions above further comprising a sequence selected from a group consisting of sequence IDs 10-22.

[0033] In some embodiments, the invention provides a therapeutic immodulator peptide complexed with metal, wherein said metal is selected from a group consisting of ferrous iron, ferric iron, zinc, copper, vanadium, ruthenium, cobalt, titanium, manganese, and calcium, or metallocene compounds containing these metals. In other embodiments, the invention provides a therapeutic immodulator peptide complexed with a glycosaminoglycan selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, and hyaluronate.

[0034] In another aspect, the invention provides a method for treating a mammal showing symptoms of immune dysfunction or perturbation comprising administering to said mammal via intramuscular, subcutaneous, parenteral, transdermal, intranasal, intravenous or intrathecal route of administration a pharmaceutical formulation comprising a therapeutically effective dose of an improved immodulator peptide according to the invention, and a pharmaceutically acceptable excipient, thereby alleviating said symptoms of dysfunction. In some embodiments, the immodulator peptide is administered in a therapeutically effective dose of peptide between about 0.01 mg/kg/day to about 50 mg/kg/day.

[0035] In another aspect, the invention provides for co-administration of a helper molecule that modulates the activity of cellular antiviral defenses. Preferably, said helper molecule is a modulator of the activity of cellular antiviral defenses, said helper selected from the group consisting of modulators of RIG-I, MDA5, MAVS, STING, IRF3, STAT1, STAT3, TBK1, PACT, LGP2, NFkappaB, DNA methylases such as 5-azacytidine or SAHA and toll-like receptors. Demethylation of DNA can derepress endogenous retroviral sequences which then, in turn, trigger cellular antiviral defense mechanisms. Preferably, the helper molecule is an agonist of RIG-I or MDA5 such as poly(I:C). Many agonists and enhancers of helper molecules are well-known in the art such as KIN-1400, a synthetic RIG-I agonist commercially available from Cayman Chemical (Ann Arbor, Mich.).

[0036] This invention envisages an in vitro assay method for measuring the biological potency of an immodulator peptide, said assay method selected from a group consisting of modulation of TLR signaling, RIG-I signaling, apoptosis or collagen synthesis in cultured cells. As will be understood by those of skill in the art, the mode of detection of a diagnostic signal will depend on the exact detection system utilized in the assay. For example, if a fluorescent detection reagent is utilized, the signal will be measured using a technology capable of quantitating the signal from the sample, such as by the use of a fluorometer. If a chemiluminescent detection system is used, then the signal will typically be detected using a luminometer. Methods for detecting signal from detection systems are well known in the art and need not be further described here.

[0037] Sequence “identity” and “homology”, as referred to herein, can be determined using BLAST, particularly BLASTp as implemented by the National Center for Biotechnology Information (NCBI), using default parameters. It will be readily apparent to a practitioner skilled in the art that sequences claimed hereunder include all homologous and trivial variants of an immodulator peptide, such as by conservative substitution, extension and deletion in their amino acid sequences. Trivial substitution variants include swapping of an amino acid with another belonging to the same class, without such substitution resulting in any significant and measurable functional improvement. “Classes” of amino acids include positively charged amino acids (arginine, lysine, histidine), negatively charged amino acids (aspartic acid, glutamic acid), aromatic amino acids (tyrosine, phenylalanine, tryptophan), branched chain amino acids (valine, leucine isoleucine) and other natural groupings such as (serine, threonine) and (asparagine, glutamine).

[0038] For testing efficacy of pharmaceutical composition containing an immodulator peptide, an effective amount of therapeutic agent is administered to a subject having a disease. In some embodiments, the agent is administered at about 0.001 to about 40 milligrams per kilogram total body weight per day (mg/kg/day). In some embodiments the agent is administered at about 0.001 to about 40 mg/kg/day, e.g., 0.01, 0.015, 0.02, 0.05, 0.1, 0.2, 0.5, 0.7, 1, 2, 4, 5, 7, 9, 10, 15, 20, 25, 30, 35 or another dose from about 0.001 mg/kg/day to about 40 mg/kg/day.

[0039] Therapeutic agents are preferably administered via oral or parenteral administration, including but not limited to intravenous (IV), intra-arterial (IA), intraperitoneal (IP), intramuscular (IM), intracardial, subcutaneous (SC), intrathoracic, intraspinal, intradermal (ID), transdermal, oral, sublingual, inhaled, and intranasal routes. IV, IP, IM, and ID administration may be by bolus or infusion administration. For SC administration, administration may be by bolus, infusion, or by implantable device, such as an implantable minipump (e.g., osmotic or mechanical minipump) or slow release implant. The agent may also be delivered in a slow release formulation adapted for IV, IP, IM, ID or SC administration. Inhaled agent is preferably delivered in discrete doses (e.g., via a metered dose inhaler adapted for protein delivery). Administration of a molecule comprising an agent via the transdermal route may be continuous or pulsatile. Administration of agents may also occur orally. For parenteral administration, compositions comprising a therapeutic agent may be in dry powder, semi-solid or liquid formulations. For parenteral administration by routes other than inhalation, the composition comprising an agent is preferably administered in a liquid formulation. Compositions comprising an agent formulation may contain additional components such as salts, buffers, bulking agents, osmolytes, antioxidants, detergents, surfactants, and other pharmaceutical excipients as are known in the art.

[0040] As will be understood by those of skill in the art, the symptoms of disease alleviated by the instant methods, as well as the methods used to measure the symptom(s) will vary, depending on the particular disease and the individual patient. All references cited in this document, including patent applications and publications cited therein, are incorporated by reference in their entirety.

Examples

Example 1. N-Terminal Modification of Peptide with Carboxylic Acids of Molecular Mass Below 500Daltons that are not Proteinogenic Amino Acids or Biotin

[0041]

TABLE-US-00002 Class Compound CAS No. MW Yld T4* Yld IM3* fatty acid oleic acid 112-80-1 282.5 44.21% fatty acid eicosapentaenoic acid 10417-94-4 302.5 66.79% fatty acid lignoceric acid 557-59-5 368.6 89.20% fatty acid decanoic acid 1002-62-6 172.2 88.67% 98.0% fatty acid docosahexanoic acid 6217-54-5 368.6 57.77% fatty acid lauric acid 143-07-7 200.3 85.14% 96.7% fatty acid 10-hydroxy-2-decenoic acid 14113-05-4 186.3 44.38% phenolic acid ferulic acid 1135-24-6 194.2 26.58% phenolic acid isoferulic acid 537-73-5 194.2 55.80% 70.2% phenolic acid Aspirin 50-78-2 180.2  56.5% phenolic acid valeroyl salicylate 64206-54-8 222.2 11.76% pentacyclic betulinic acid 472-15-1 456.7  .sup. <1% triterpenoid anthraquinone Rhein 478-43-3 284.2 50.95% anthraquinone Diacerein 13939-02-1 368.3  43.2% Xanthone 2,7-dichlorodihydro- 4091-99-0 487.3  91.2% fluorescein diacetate proprionic acid (s)-ketoprofen 22161-81-5 254.3 77.86% proprionic acid Ibuprofen 15687-27-1 206.3 93.42% 98.0% carboxylic acid trans-cinnamic acid 140-10-3 148.2 93.12% 81.5% carboxylic acid (s)-(−)-perillic acid 23635-14-5 166.2 27.96% carboxylic acid fenofibric acid 42017-89-0 318.8 85.67% 99.9% indoleacetic acid Indomethacin 53-86-1 357.8  87.5%  85.2%# pentanoic acid valproic acid 1069-66-5 144.2 91.43% 84.9% alkynoic acid 2-hexyl-pentynoic acid 96017-59-3 182.3 85.1% indolylcarboxylic RG-108 48208-26-0 334.3  74.3%@ Retinoid all-trans retinoic acid 302-79-4 300.4  13.1% Rexinoid Bexarotene 153559-49-0 348.5 97.09% 94.4% Covalent terminal conjugation of carboxylic acids to peptides. *Percent yield of correct species by MS for T4 (tetrapeptide) and IM3 immodulator peptide (industrially desirable >80% shown in bold type); #lost p-chlorophenone group (incorrect product); @indole core oxidized by Arg protecting groups (incorrect product);

[0042] N-terminal modification of peptides with biotin, or fatty acids such as myristic and palmitic acids, has been commonly disclosed in the literature. Other types of carboxylic acids have rarely, if ever, been used in this way. This example shows the difficulty in predicting success for this type of modification for a previously untried carboxylic acid without doing the actual experiment. The data in this example disclose, amongst other facts: (i) coupling to carboxylic acids (that are not proteinogenic amino acids or biotin) using normal peptide synthesis conditions; (ii) surprising results showing that yields of correctly coupled product (as ascertained by mass spectroscopy analysis) varied greatly, even within the same class of compound; and (iii) consistent results for small molecules attached by this method to immodulator peptide IM3 (SEQ ID NO: 5) or a generic D-tetrapeptide dLys-dAsp-dLys-dPro (SEQ ID NO: 9), with similar efficiencies of coupling to either peptide, thereby demonstrating the generality of the method. Peptides were synthesized according to a common Fmoc/tBu solid phase synthesis strategy well-known in the art. Synthesis may be manual of automated. After the peptide synthesis the resin was divided into batches of 20 umol. Each batch was treated with one of the organic compounds specified in Table x1B below. The coupling was carried out using 2 equivalents of the compound, 2.4 equivalents of activator HATU or HCTU, and 4 equivalents of NMM base. The reaction mixture was renewed after 2 hrs reaction time and allowed to react another 4 hrs or overnight. After washing the resin several times with DMF, and subsequently with DCM, the batches were dried. For the cleavage of the peptides from the resin the resins were treated with 1% DTT, 2% water and 3% TIPS in TFA for 3.5 hrs. The cleavage solution was separated from the resin and treated with diethylether/n-pentane (1:1). The resulting precipitate was centrifuged and the pellet washed three times in the same DEE/pentane mixture. The recovered peptide was air dried and stored at −20 degrees C. or further purified by HPLC using a 0-50% acetonitrile gradient, 0.1% trifuoroacetic acid (20 min). The results of the above conjugation experiments show that, both inter-class and intra-class, there is wide variation in conjugation efficiency from compound to compound. As the practicality and cost of synthesis can be dramatically affected when product yield is low, it is therefore not obvious that any untested carboxylic acid should be assumed to be a good candidate for this type of peptide modification. The use of most of the compounds tested here has never been reported for this kind of peptide modification. It appears that that chance of practical success (>80% correct yield, for instance) for each instantiation of this technology is less than 50% until tested.

Example 2. Metal binding

[0043] The following peptides were used in experiments involving streptavidin-coated plates or Alexa488-labeled streptavidin reagent (N-terminally biotinylated versions in some instances):

TABLE-US-00003 IM1 (KNGFYHSRQCETSMDGEAGLCW) IM2 (KHGLYNLKQCKMSLNGQRGECW) IM3 (KKGFYKKKQCRPSKGRKRGFCW) IM4 (RNGNFHPKQCHPALDGQRGKCW) IM5 (RKGFYKRKQCKPSRGRKRGICW) IM6 (HRGFYRKRQCRSSQGQRRGPCW) IM3N1 (RGVTEDYLRLETLVQKVVSKGFYKKKQCRPSKGRKRGFCW) IM3N2 (TGPSESYLQLEELVKQVVSKGFYKKKQCRPSKGRKRGFCW) IM3AVD: KKGFYKKKQCRPSKGRKRGFCWAVD IM3dAVD: KKGFYKKKQCRPSKGRKRGFCW(dA)VD IM3FVS: KKGFYKKKQCRPSKGRKRGFCWFVS

[0044] Experiment x2A. Metal-immodulator peptide IM3N1 binding (detection by ELISA) The NTA-metal-affinity ELISA assay was performed using 96-well NTA-metal-coated plates readily available from commercial sources such as Qiagen (Germantown, MD). The NTA was charged with ferrous or ferric iron, zinc, copper, calcium, vanadium, ruthenium, cobalt, titanium, manganese or other metals. 400 ng of immodulator peptide IM3N1 in PBS buffer was added per plate. After incubation at room temperature for 60 minutes, excess peptide was washed off and peptide was detected by ELISA using an anti-immodulator rabbit primary antibody (A2501) and horseradish peroxidase conjugated to a secondary anti-rabbit antibody (arbitrary units) * vs EDTA control: [0045] EDTA (no metal): 0.047±0.001 [0046] Fe2+(Ferrous): 0.844±0.026 (p<0.001*) [0047] Fe3+(Ferric): 0.848±0.011 (p<0.001*) [0048] Zn2+(Zinc): 0.194±0.089 (p<0.05*) [0049] Cu2+(Copper): 0.229±0.086 (p<0.05*)

[0050] Experiment x2B. Metal-immodulator peptide binding (detection by fluorescence) NTA-metal-affinity fluorescence assay was performed using 96-well NTA-metal-coated plates readily available from commercial sources such as Qiagen (Germantown, MD). NTA was charged with ferrous or ferric iron, zinc, copper, calcium, vanadium, ruthenium, cobalt, titanium, manganese or other metals. 400 ng of FITC-labeled immodulator peptide (or biotinylated immodulator peptide complexed to Alexa-488-labeled streptavidin) in PBS buffer was added per plate. After incubation at room temperature for 60 minutes, excess peptide was washed off and peptide was detected by measuring fluorescence (excitation 490 nm, emmission 525 nm) in a fluorometer. [0051] (a) Using FITC-labeled IM3 peptide (1 ug/well) in the fluorescent binding assay: [0052] EDTA-treated control: 2,251±1,186 [0053] Ferrous iron: 170,441±11,264 (p<0.001)* [0054] Copper: 13,644±5566 (p<0.05)* [0055] Calcium: 48,822±11,990 (p<0.01)* [0056] p values are relative to EDTA-treated control

[0057] (b) Competition binding assay to ferrous iron, calcium, copper and zinc-charged NTA-coated plates using FITC-labeled IM3 peptide (0.5 uM) and 8X (4 uM) unlabelled competitor peptides IM3N1, IM3N2 (*all comparisons to PBS control are p<0.01); # fluorescent value of binding to calcium was set at 100.

TABLE-US-00004 Metal Compet: PBS Compet: IM3N1 Compet: IM3N2 Calcium # 100.0 ± 1.9  14.6 ± 1.4* 12.9 ± 1.2* Ferrous iron 85.6 ± 5.8 20.4 ± 1.0* 25.2 ± 9.5* Copper 53.0 ± 0.6  9.2 ± 1.5*  5.1 ± 1.0* Zinc 85.9 ± 5.5 15.7 ± 1.3* 13.4 ± 1.6*

[0058] (c) Alexa488-SA-labelled immodulator peptide IM3 (400 ng/well) bound to NTA-coated plate charged with the indicated metals: #fluorescent value of binding to ferrous iron was set at 100.*p<0.01 relative to control. Ferrous iron # (100±2.6*); Control (no metal): 6.4±3.1; Ferric iron: 94.1±6.6*; Calcium: 47.6±3.7*; Vanadium: 53.9±5.5*; Manganese: 54.4±11.2*; Cobalt: 25.4±2.3*; Copper: 58.4±6.4*; Zinc: 71.4±8.3*; Ruthenium: 67.8±8.3*; (d) Alexa488-SA-labelled immodulator peptide IM1, IM2, IM3, IM4, IM5, IM6 (400 ng/well) were bound to NTA-coated plate charged with the indicated metals: # background (no peptide) control set to 1.0;*p<0.05, **p<0.01 vs “no peptide”

TABLE-US-00005 Peptide Ferrous iron Calcium Copper Zinc IM1 1.2 ± 0.3 1.1 ± 0.1  3.0 ± 0.1** 1.1 ± 0.2 IM2 1.4 ± 0.3 1.7 ± 0.2* 3.6 ± 0.1** 1.1 ± 0.1 IM3  24.7 ± 1.6** 28.7 ± 0.7** 6.2 ± 0.2**  32.0 ± 0.7** IM4 2.8 ± 1.4 1.0 ± 0.1  5.7 ± 0.1** 1.2 ± 0.2 IM5  20.5 ± 1.5** 23.4 ± 0.8** 5.6 ± 0.1**  23.6 ± 0.1** IM6  20.6 ± 0.8** 15.1 ± 1.9** 2.4 ± 2.4**  8.4 ± 0.4** No 1.0 ± 0.1 1.0 ± 0.0  1.0 ± 0.2  1.0 ± 0.1 peptide #

[0059] Experiment x2C. Binary Peptide-Metal Complexes.

[0060] Preparation of binary peptide-metal complexes in PBS buffer was performed by incubating 4 mg of IM3N1 peptide in 5 mM metal solution at room temperature for 60 min. The mixture was desalted using a GT-100 SpinOUT column (G-Biosciences Inc, St. Louis, Mo.) equilibrated in PBS buffer. Recovery of peptide was 30-35% for zinc and iron complexes. In the case of iron, confirmation of metal within the complex per mg peptide was made using a commercial kit for iron assay. Additionally, peptide-metallocene complex were prepared using IM3 peptide and a ferrocene derivative, ferroquine (purchased from Sigma-Aldrich, St. Louis, Mo.).

[0061] Experiment x2E. Immodulator peptide variants showing enhanced metal binding.

[0062] Immodulator peptides were extended C-terminally by three amino acids, to include a valine residue that is highly conserved in the “thyroglobulin type 1 motif′. The reasoning behind this attempt is that a longer conserved sequence might lead to a tighter fold overall, which might in turn result in increased metal-binding avidity.

[0063] (b) Strength of binding to iron was evaluated by plate assay.

[0064] Experiments were conducted by the procedure described above. Ferric iron-charged NTA-coated 96-well plates were used as described. 1 ug/well immodulator peptide complexed to Alexa-488-labeled streptavidin in PBS buffer was added (60 min at room temperature). The plate was washed and counted in a standard fluorometer (excitation/emission: 485/525 nm). The results (fluorescence) show improved iron-binding by the three peptides extended C-terminally by the AVD, (dA)VD, and FVS tripeptides. ** p<0.01 vs IM3 control IM3: 200±29; IM4: 45±37**; IM5: 130±77; IM3AVD: 434±29**; IM3dAVD: 418±28**; IM3FVS: 371±33**

[0065] (c) Competition assay. The same procedure was used as the preceding experiment, except that 375 ng/well labeled IM3 peptide was used, plus 10x of the indicated unlabeled peptide. ‘No competing peptide’ control value was set to 100. * p<0.05 vs “no peptide” control Competing Peptide/Fluorescence None: 100±38; IM4: 125±26; IM3: 63±27*; IM5: 40±14*; IM3AVD: 10±28*

[0066] (d) Competition assay. The same procedure was used as the preceding experiment, except that labeled IM3AVD peptide was used. ** p<0.01 vs “no competing peptide” control None: 100±9; IM4: 144±44; IM3: 35±16**; IM5: 30±19**; IM3AVD: 9±19**

[0067] (e) The same procedure was used as the preceding experiment, except that each peptide was labeled with Alexa-488-labeled streptavidin in PBS buffer and used separately. Binding to IM3 was set to =100. * p<0.05, ** p<0.01 vs IM3 control. (Labeled Peptide/Fluorescence) IM1: 1.8±0.4′; IM2: 3.0±1.8**; IM3: 100.0±16.8; IM4: 7.2±1.7*; IM5: 86.7±6.4; IM6: 85.9±14.9; IM3AVD:137.7±11.8*; IM3dAVD:147.4±22.9*; IM3FVS: 101.5±10.5. The results of the above experiments show that when the IM3 sequence is extended C-terminally by the AVD tripeptide, iron is bound more tightly.

Example 3. Additional Novel Properties of Immodulator Peptides

[0068] Experiment x3A. Binding of immodulator peptides to AA111-228 DNA-binding domain of RXR-alpha (RXRa-DBD). 1 ug/well recombinant RXRa-DBD (Abcam, Cambridge, Mass.) was adsorbed to wells of a 96-well plate for 60 minutes at RT, then blocked with 200 uL 1% BSA in PBS buffer overnight. Plate was washed and 800 ng/well Streptavidin-Alexa 488 conjugate (SA488)-labelled IM peptide was added. The plate was incubated for 60 min at room temperature, washed and counted in a standard fluorometer (excitation/emission 485/525 nm). Background (buffer alone) was subtracted. The results are shown in Table x3A. They show that IM3, IM5 and IM6 bind RXRa-DBD. *p<0.05, **p<0.01 vs no peptide; (Peptide/Fluorescence) IM1: 670±85*; IM2: 2±38; IM3: 6,572±129**; IM4: 27±31; IM5: 6,802±336**; IM6: 587±192*; No peptide: 8±232.

[0069] Experiment x38. Binding of immodulator peptides to DNA-binding domain (DBD) of RXR-alpha, with or without ferric iron. 100 ng/well recombinant RXRa-DBD (Abcam, Cambridge, Mass.) was adsorbed to wells of a 96-well plate for 60 minutes at RT, then blocked with 200 uL 1% BSA in PBS buffer overnight. Plate was washed and 800 ng/well Streptavidin-Alexa 488 conjugate (SA488)-labelled IM peptide was added, with or without ferric chloride (5 uM) or 5-fold excess RXRa-DBD. The plate was incubated for 2 hours at room temperature, washed and counted in a standard fluorometer (excitation/emission 485/525 nm). The results are shown in Table x3B. They show that IM3 and IM5 bind RXRa-DBD, iron stimulates this binding, and cold RXRa-DBD competes against it.

TABLE-US-00006 Peptide FeCl3 excess RXRa-DBD Fluorescence None None None 88.0 ± 9.8.sup.  IM3 None None  .sup. 687.3 ± 55.4**## IM3 5 uM None 1161.3 ± 78.5   IM3 5 uM 5X 837.0 ± 30.5 # IM5 None None 228.0 ± 44.9*  IM5 5 uM None 552.7 ± 86.8.sup.  IM5 5 uM 5X 303.0 ± 68.3 # *p < 0.05, **p < 0.01 vs no peptide; # p < 0.05, ##p < 0.01 vs binding with iron

[0070] Experiment x3C. Binding of immodulator peptides to full-length huNur77. 400 ng/well recombinant Nur77 (Abcam, Cambridge, Mass.) was adsorbed to wells of a 96-well plate for 90 minutes at RT, then blocked with 200 uL 1% BSA in PBS buffer overnight. Plate was washed and 333 ng/well Streptavidin-Alexa 488 conjugate (SA488)-labelled IM3, IM4 or no peptide was added. The plate was incubated for 60 min at room temperature, washed and counted in a standard fluorometer (excitation/emission 485/525 nm). Background (buffer alone) was subtracted. The results are shown in Table x3C. They show that IM3 but not IM4 binds Nur77. **p<0.01 vs

[0071] IM3+Nur77; #p<0.01 vs no peptide+no Nur77 control;

TABLE-US-00007 Peptide Nur77 Fluorescence None None 63 ± 34 None 400 ng  98 ± 59** IM3 None 59 ± 17 IM3 400 ng  488 ± 38 # IM4 None 112 ± 24  IM4 400 ng  70 ± 48**

[0072] Experiment x3D. Binding of immodulator peptides to glycosaminoglycan, heparin. 1 ug Streptavidin-Alexa 488 conjugate (SA488)-labelled IM peptide in PBS buffer was loaded on a spin column packed with 0.2 ml bed volume heparin-agarose (G-Biosciences, St. Louis, Mo.). The column was centrifuged for 2 min at 2,000G and the flow-through was collected. The column was then washed with 0.3 ml 1% BSA in PBS buffer and the column spin repeated. Flowthrough and wash were pooled and duplicate 20 uL aliquots counted in a standard fluorometer (excitation/emission 485/525 nm). Results are expressed as percentage of input counts that bound the heparin-agarose column, versus flowthrough. Percent counts bound to heparin-agarose resin are shown below. IM3, IM5 and IM6 peptides bind heparin-agarose in this assay. The other 3 peptides do not.

TABLE-US-00008 Peptide Percent retained Percent Flowthrough None 0 100.0 IM1 0 100.0 IM2 0 100.0 IM3 97.7 2.3 IM4 0 100.0 IM5 96.4 3.6 IM6 98.0 2.0

[0073] Example 4. Modulation of key immunological pathways using immodulator peptides. Immune modulation functions of immodulator peptides were measured using the monocyte reporter cell line THP1-Dual (Invivogen, San Diego, Calif.), which provides convenient readouts for STING and NF-kappa-B pathways. Optionally, succinic acid, cytokine and chemokine production were also measured. THP1-Dual cell line was obtained from Invivogen. Cells were grown in a T-75 flask in RPMI Medium containing 20% fetal bovine serum, blasticidin (10 mg/mL), zeocin (100 mg/mL) and penicillin-streptomycin at 370C in a humidified, 5% CO.sub.2 incubator. Cells (100 ml, 8,000 cells/well) were plated in a 96-well plate and incubated overnight at 370C in a humidified, 5% CO2 incubator. Next day, 10 ml/well of compounds were added (quadruplicate wells). After 24 hour incubation with the compound, supernatants were harvested and stored at 4 0C prior to assay.

[0074] Experiment x4A. TLR- and RIG-I-stimulating activity of immodulator peptides in THP1-Dual monocytes. Each cell supernatant was assayed in quadruplicate. 20 uL of supernatant was used to measure NF-kappa-B (NFkB) and STING activity using reagents QuantiBlu and QuantiLuc respectively, according to the manufacturer's recommendations (Invivogen, San Diego, Calif.). Succinic acid was measured by using a kit from Megazyme Inc (Cambrigdge, MA). The results of this experiment are shown in Tables x4A-D below. Control value of activity was set to 100 in each assay. The data show that the stimulating activity of various immodulator peptides is influenced by specific extension sequences and by the kind of N-terminally conjugated carboxylic acid. PAM3C=25 ng/ml PAM3CSK4 (TLR1/2 inducer); Poly(I:C)=10 ug/ml (TLR3 inducer); PAM2C=25 ng/ml PAM2CSK4 (TLR2/6 inducer); KIN14=4 uM KIN-1400 (RIG-I inducer); cytB=1 uM cytosporone B (Nur77 inducer); C-178=1 uM STING inhibitor; NFkBi=50 ug/ml NFkB inhibitor peptide; SB2021=10 uM p38MAPK inhibitor; *p<0.05, **p<0.01 vs 1M3 control; # p<0.01 vs “no inducer” control; SUCC=succinic acid; nd=not done; Peptide was added to cells at 400 nM.

TABLE-US-00009 Peptide Inducer Modulator NFkB STING SUCC None 100 100  Nd IM3 None   94**  91** nd PAM3C   326 # 342 # IM3 PAM3C 575 1474  100 IM3 PAM3C cytB 576 1318** 95.4 IM3 PAM3C C-178 561  566** 95.3 IM3 PAM3C NFkBi  458** 1294** 101.5 IM3 PAM3C SB2021  296**  274** 95.8

TABLE-US-00010 Peptide Reagent NFkB STING SUCC PAM2C 118.7** 144.5** 121.5* IM3 PAM2C 100 100 100 IM3 PAM2C cytB 99.8 96.5 85.1* IM3 PAM2C C-178 108.0** 62.0** 96.1 IM3 PAM2C NFkBi 118.7* 119.1* 100.1 IM3 PAM2C SB2021 65.4** 18.9** 93.4

[0075] Peptides were added to cells at 400 nM. Inducer was KIN1400 (RIG-I agonist).

TABLE-US-00011 Peptide Reagent NFkB STING SUCC 90.1 84.3** 100.1 IM3 100 100 100 IM3 cytB 103.5 87.5* 96.1 IM3 C-178 98.1 81.1** 103.2 IM3 NFkBi 104.4 105.0 99.2 IM3 SB2021 131.3** 75.6** 131.6*

Example 5. Positive effects of ferric iron and covalent modification of peptide on the systemic efficacy of nephrilin IM3N1 peptide in burn trauma

[0076] Experiment x5A. Nephrilin peptide (IM3N1) is a designed inhibitor of Rictor complex (also known as mTORC2), an evolutionarily conserved assembly believed to modulate responses to cellular stress. We previously demonstrated the ability of nephrilin peptide to suppress neuroinflammation, loss of body mass, glycemic control and kidney function in a rat scald model, as well as sepsis mortality in a mouse model. This study explores the effect of nephrilin plus iron formulations on clinically relevant outcomes in the rat scald model. Animals were treated with nephrilin by subcutaneous bolus injection on days 1-7 post-burn. Equimolar ferric iron in the formulation improved the positive systemic effects of nephrilin on kidney function, glycemic control, oxidative stress, early hyperinflammation, late inflammasome activation, hyperangiogenesis and body mass, all variables previously shown to bear upon clinically relevant burn injury outcomes. The rat scald model and measurement of clinically relevant variables are disclosed in U.S. Pat. No. 10,369,191. Adult Sprague Dawley rats of both sexes (250-300 gm, Charles River Laboratories, Wilmington, Mass., USA) were injected with nephrilin peptide plus or minus equimolar metal (zinc, ferrous iron or ferric iron) once daily by subcutaneous bolus injection, days 1-7; Treatment group sizes were (n=8) for each sex unless otherwise indicated: group S=sham-treated; group B=burn+vehicle; group N1=burn+4 mg/kg nephrilin; group N1/Zn2=burn+4 mg/kg nephrilin/zinc chloride; group N1/Fe2=burn+4 mg/kg nephrilin/ferrous sulphate; groups N1/Fe3 (2, 4 or 8)=burn+2, 4, or 8 mg/kg nephrilin/equimolar ferric chloride. The first dose was administered after completion of the scald procedure. Injection volume was 400 uL. Control animals received the same volume of vehicle.

[0077] Male rats: Ferric iron improves efficacy of nephrilin peptide Table x5A1 shows the results obtained when male rats in the scald model were exposed to vehicle, 4 mg/kg nephrilin and 4 mg/kg nephrilin complexed with either ferric iron, ferrous iron or zinc. To make global comparisons of treatment efficacy, we converted values to z-scores. All scores within an efficacy class were averaged, allowing allocation of equal weight to efficacy classes. A composite z-score (average z score across all 7 effect classes) is shown in the last row of the table. This allows direct comparison of efficacy across treatment groups. The sham treatment involves manipulating the animals exactly as in the burn group, but without the burn treatment. The efficacy value for the sham (0.82) is thus the positive control. The negative control is the Burn+vehicle group (-0.61). The results show that ferric iron supplementation is superior to the other metals (efficacy of 0.27 vs 0.01 and -0.19 for zinc and ferrous iron, respectively) in improving the efficacy of nephrilin. Compared to vehicle or nephrilin treatment alone (-0.31), nephrilin with ferric iron (0.27) is at least twice as efficacious overall. Male rats in the scald model were exposed to vehicle, 2, 4 and 8 mg/kg nephrilin complexed with ferric iron. The results show that 2 mg/kg (0.17) and 4 mg/kg (0.27) dose levels were superior to 8 mg/kg (-0.37).

TABLE-US-00012 TABLE x5A1 Efficacy in male rats. Treatment groups (n = 8) are described in Methods. Effect classes - 1: early inflammation (24-hr plasma IL-6 pg/ml); 2: 14-day plasma inflammasome markers (2a = IL18 pg/ml; 2b = IL1b pg/ml); 3: 14-day plasma hyperangiogenesis markers (3a = VEGF-A pg/ml; 3b = CCL5 pg/ml; 3c = CXCL5 pg/ml; 3d = PctRedPix); 4: glycemic control, 14-day GTT (AUC mg .Math. dL .Math. hr); 5: kidney function, 14-day plasma (creatinine mg/dL); 6: systemic oxidative stress, 14-day plasma (OHDG pg/ml); 7: weight loss (slope). S B N1 N1/Zn2 N1/Fe2 N1/Fe3 N1/Fe3 N1/Fe3 x5A (Sham) (vehicle) 4 mg/kg 4 mg/kg 4 mg/kg 2 mg/kg 4 mg/kg 8 mg/kg 1  31.4 ± 12.0*  62.3 ± 16.9 50.6 ± 6.1  43.4 ± 13.6* 49.1 ± 13.1 38.5 ± 27.8  41.4 ± 11.2* 48.5 ± 18.5 2a 125.2 ± 45.5*  203.8 ± 61.8 178.1 ± 19.3 133.3 ± 25.1  157.7 ± 70.7  220.4 ± 32.1  119.1 ± 15.4* 148.2 ± 61.7  2b 41.9 ± 12.3*  266.8 ± 185.2 124.3 ± 132  36.7 ± 9.5* 97.4 ± 86.6 52.9 ± 7.8* 36.6 ± 7.8* 254.5 ± 291.9 3a 50.4 ± 10.2*  67.4 ± 17.4  56.1 ± 11.7 38.3 ± 8.8* 41.2 ± 6.8*  37.8 ± 15.3*  48.1 ± 10.9* 43.3 ± 8.1* 3b 218.1 ± 41.8*  664.8 ± 42.8 609.7 ± 283  321.1 ± 96   249.8 ± 73*.sup.  248.3 ± 29.7*   192.1 ± 26.3*#& 301.2 ± 74.9  3c 763 ± 229* 2009 ± 745 1910 ± 380 1154 ± 224*  939 ± 300*#    855 ± 233*#& 1038 ± 495* 1435 ± 445  3d n/a 20.01 ± 1.37 16.51 ± 3.02  9.74 ± 5.79* 11.79 ± 5.6*   6.53 ± 3.16*    8.65 ± 3.35*#&  18.3 ± 11.83 4  78.4 ± 43.4* 157.3 ± 69.8 139.3 ± 48.6 215.6 ± 124.sup.  120.6 ± 65.4  136.4 ± 69.2   94.3 ± 25.4*  93.8 ± 37.2* 5  0.23 ± 0.03*  0.38 ± 0.18  0.34 ± 0.13 0.26 ± 0.12 0.38 ± 0.16 0.31 ± 0.09    0.18 ± 0.11*#& 0.38 ± 0.12 6  479 ± 116* 665 ± 78  585 ± 137  454 ± 76*#  455 ± 71*#    374 ± 187*#&   423 ± 79*#& 761 ± 89  7  5.58 ± 1.00*  2.34 ± 1.03  2.32 ± 1.01 1.66 ± 1.08 2.65 ± 0.56 2.99 ± 0.53 2.71 ± 1.62 2.50 ± 0.55 Avg 0.82 ± 0.58* −0.61 ± 0.47 −0.31 ± 0.35  0.01 ± 0.62* −0.19 ± 0.52*  0.17 ± 0.40*    0.27 ± 0.60*#& −0.37 ± 0.45  *p < 0.05 vs B group; #p < 0.05 vs N1 group; &p < 0.05 vs 8 mg/kg group.

[0078] Experiment x58. Effect of N-terminal modifications on the efficacy of nephrilin IM3N1 peptide in the rat scald model. In this study, five nephrilin-iron binary complexes were compared: IM3N1 (unmodified) and the following: IM3N1v1p (valproic acid), IM3N1dec (decanoic acid), IM3N1fen (fenofibric acid), and IM3N1ibu (ibuprofen). Adult male Sprague Dawley rats (300 gm, Charles River Laboratories, Wilmington, Mass., USA) were injected with peptides formulated with equimolar metal ferric iron once daily by subcutaneous bolus injection, days 1-7.

TABLE-US-00013 Burn + Burn + Burn + Burn + Burn + Burn + x5B Sham Vehicle IM3N1 IM3N1vlp IM3N1dec IM3N1fen IM3N1ibu 1  31.4 ± 12.0*  62.3 ± 16.9 38.5 ± 27.8 24.4 ± 19.2* 35.4 ± 22.9 54.8 ± 29.6  68.9 ± 20.4 2a 125.2 ± 45.5*  203.8 ± 61.8 220.4 ± 32.1  142.4 ± 53.2  170.9 ± 25.2  183.7 ± 46.5  119.7 ± 50.6* 2b 41.9 ± 12.3*  266.8 ± 185.2 52.9 ± 7.8* 62.8 ± 17.2*  53.2 ± 11.6* 63.2 ± 14.9* 43.2 ± 4.4* 3a 50.4 ± 10.2*  67.4 ± 17.4  37.8 ± 15.3* 44.3 ± 11.5* 29.9 ± 8.7* 38.3 ± 11.5*  32.7 ± 15.3* 3b 218.1 ± 41.8*  664.8 ± 42.8 248.3 ± 29.7* 220.2 ± 43.4*  243.1 ± 52.2* 285.2 ± 104.3* 202.1 ± 88.2* 3c 763 ± 229* 2009 ± 745  855 ± 233* 717 ± 324*  717 ± 285* 774 ± 232*  589 ± 377* 3d n/a 20.01 ± 1.37  6.53 ± 3.16*  1.46 ± 0.87*#  4.83 ± 3.37* 5.30 ± 2.68* 3.17 ± 1.5* 4  78.4 ± 43.4* 157.3 ± 69.8 136.4 ± 69.2  57.5 ± 40*#  112.0 ± 55.9  114.9 ± 59.7  89.8 ± 71.6 5  0.23 ± 0.03*  0.38 ± 0.18 0.31 ± 0.09  0.17 ± 0.03*# 0.33 ± 0.11 0.29 ± 0.09   0.24 ± 0.07* 6  479 ± 116* 665 ± 78  374 ± 187* 360 ± 121* 597 ± 115 870 ± 265  712 ± 164 7  5.58 ± 1.00*  2.23 ± 1.08 2.99 ± 0.53  3.74 ± 0.31*# 3.40 ± 0.75 3.32 ± 0.76  3.55 ± 1.43

[0079] Treatment group sizes were (n=6) for each group: group S=sham-treated; group B=burn+vehicle; group N1=burn+2 mg/kg nephrilin IM3N1; group N1V=burn+2 mg/kg IM3N1vIp; group N1D=burn+2 mg/kg IM3N1dec; group N1F=burn+2 mg/kg IM3N1fen; group N11=burn+2 mg/kg IM3N1ibu. The first dose was administered after completion of the scald procedure. Injection volume was 400 uL. Control animals received the same volume of vehicle. Rat scald model was performed and measurements taken as described in Experiment x5A. The Table x5B (above) shows the results obtained when male rats in the scald model were exposed to vehicle, or 2 mg/kg peptide complexed with ferric iron: (V) valproic acid; (D) decanoic acid; (F) fenofibric acid; (I) ibuprofen; for left column, see legend to Table x5A; * p<0.05 vs burn+vehicle; # p<0.05 vs burn+IM3N1 group. The results show that 2 mg/kg IM3N1v1p dose levels were superior to 2 mg/kg unmodified IM3N1 peptide.

Example 7. Transcriptional re-programming in rat CNS two weeks after burn trauma: the impact of nephrilin IM3N1 peptide treatment on the expression of key genes associated with oxidative stress

[0080] Survivors of severe burns suffer lifetime neuroinflammatory consequences manifested by higher incidence of major depression and neurodegenerative disease. In a scald model, nephrilin IM3N1 peptide has previously been shown to protect rats from loss of lean body mass, kidney function and glycemic control, complications that endure in burn patient populations. Nephrilin's mechanism of action appears to involve protection from excessive oxidative stress. Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) amplification of transcripts in total RNA extracted from dorsal root ganglia (DRG) of male rats 14 days after exposure to thermal insult, we query the relative levels of expression of 34 genes believed to be associated with oxidative stress biology in the CNS. We use these data to explore the central role of oxidative stress in astrogliosis, immunosuppression and mitochondrial homeostasis. This subset analysis of DRG from a previously published study (n=3 per group) is associated with the following effects in the study (Table x7A):

TABLE-US-00014 TABLE x7A BURN + BURN + SHAM VEHICLE IM3N1 Lean Body Mass 343.6 ± 15.7*  304.9 ± 9.2  328.5 ± 5.1*  (DEXA) Glycemic Control 44.7 ± 19.0** 117.0 ± 19.1 69.0 ± 18.1* (GTT AUC mg .Math. dL .Math. hr) Kidney Function 1.21 ± 0.23*   0.58 ± 0.18 1.29 ± 0.32* (eGFR, calculated) Urinary 8-isoprostate 5.02 ± 2.91** 26.44 ± 1.60  4.53 ± 1.21** (ng/pg cystatin)

[0081] The Table below shows the primers used in the RT-PCR. Rats that received nephrilin IM3N1 treatment (4 mg/kg by subcutaneous bolus injection once daily for seven days after scald injury) showed significantly reduced elevations in gene expression of key genes. In this study, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) amplification of transcripts in total RNA extracted from dorsal root ganglia of male rats 14 days after exposure to thermal insult, the relative levels of expression of 34 genes believed to be associated with oxidative stress biology in the CNS were queried, with an emphasis on astrogliosis, immunosuppression and mitochondrial homeostasis. The 34 genes studied were selected based on their known implication in the above processes, as described in the scientific literature. Dorsal root ganglia (DRG) were dissected from male adult Sprague Dawley rats (300 gm, Charles River Laboratories, Wilmington, Mass., USA), three randomly selected animals in each treatment group were used (Group 1:sham; Group 2: burn+vehicle; Group 3: burn+IM3N1) and total RNA was extracted from each pool using the Rneasy Midi Kit (Qiagen, Germantown, MD). Yield was−30 ug RNA per pool and A260/A280 ratios were between 1.87-2.04 in all cases. The high quality of each RNA prep was further confirmed by electrophoresis (using Eukaryote Total RNA Nano). For quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) RNAs were diluted in RNase/DNase free water and aliquoted into wells in triplicate. Approximately 100 ng of RNA was used per well. A one step qPCR method was performed using Luna Universal One-Step RT-qPCR kit (New England Biolabs, Ipswich, Mass.) containing reverse transcriptase enzyme mix. Primer pairs for each gene were synthesized for the SYBR assay. The following standard qPCR cycling conditions were used: 55C for 10′ (for RT), 95C for 1′ followed by 40 cycles at 95C for 10s, 58C for 30s. Background was set at 3-10 cycles and the threshold was set at 0.02 for all runs. Ct values were collected and analyzed using the “delta-delta Ct” method. Transcript data for each gene are shown relative to GAPDH, a house-keeping gene. Sham group value was set to 1. Gene groups: [A] astrocyte activation; [B] MDSC-mediated immunosuppression; [C] Mitochondrial homeostasis; [D] Downstream effects of oxidative stress;

TABLE-US-00015 Group 1 Group 2 Group 3 Mean SD p vs gp2 Mean SD Mean SD p vs gp2 [A] GFAP 1.03 0.05 0.0073 6.08 0.76 2.40 0.38 0.0052 CTGF 0.94 0.09 0.0062 3.14 0.36 1.61 0.16 0.0086 AQP4 1.06 0.09 0.0106 9.18 1.47 0.59 0.12 0.0093 Pi16 1.10 0.11 0.0004 7.90 0.36 2.40 0.12 0.0005 GALANIN 0.98 0.07 0.0078 31.73 4.72 18.50 2.14 0.0250 NPY 1.02 0.07 0.0068 47.46 6.66 29.50 3.67 0.0245 S100A9 1.00 0.07 0.0132 8.85 1.58 5.01 0.56 0.0401 [B] SLC7A11 1.02 0.09 0.0045 3.41 0.35 4.78 0.41 NS NRF2 1.00 0.06 0.0138 1.27 0.09 1.29 0.12 NS ARG1 0.99 0.04 0.0000 3.01 0.08 3.09 0.38 NS CD63 1.07 0.06 0.0309 1.95 0.29 1.88 0.19 NS CCL2 0.95 0.07 0.0006 2.62 0.04 3.17 0.23 NS [C] TFAM 1.09 0.12 0.0017 1.81 0.09 1.14 0.06 0.0011 UCP2 1.09 0.08 0.0130 1.73 0.18 1.22 0.05 0.0295 BOK 0.97 0.05 0.0048 1.92 0.15 1.10 0.07 0.0041 RAC1 1.05 0.06 0.0034 1.66 0.06 1.00 0.15 0.0088 NOX2 1.10 0.11 0.0002 2.59 0.06 2.07 0.09 0.0017 NOX4 0.97 0.04 0.0031 6.53 0.55 6.57 0.49 NS OXR1 0.98 0.12 0.6475 1.02 0.21 1.04 0.10 NS HCRTR1 1.09 0.10 0.0343 3.20 0.72 1.42 0.19 0.0423 SHC1 1.10 0.13 0.0053 1.82 0.05 2.34 0.23 NS GLUL 1.06 0.04 0.0018 1.64 0.07 1.53 0.06 NS SLC23A2 1.08 0.14 0.0356 1.43 0.13 1.47 0.13 NS STEAP4 1.06 0.09 0.0032 1.53 0.05 1.77 0.23 NS PINK1 1.02 0.14 0.0474 1.47 0.02 1.35 0.02 NS MFN2 1.02 0.11 0.0138 1.54 0.14 1.48 0.11 NS FIS1 0.98 0.04 0.1934 0.93 0.03 0.90 0.14 NS PPARGC1A 1.03 0.16 0.8383 1.13 0.22 1.00 0.09 NS ACMSD 0.98 0.03 0.6012 0.95 0.09 0.97 0.12 NS [D] PLSCR1 1.01 0.12 0.0007 2.27 0.16 2.30 0.32 NS BACE1 0.99 0.08 0.0004 1.75 0.10 1.42 0.15 NS SLC12A5 1.00 0.07 0.0354 1.74 0.27 1.38 0.15 NS SLC12A2 1.03 0.14 0.0484 1.37 0.02 1.59 0.15 NS

Example 8. Post-Burn Wound Healing Study with Peptide IM3v1p

[0082] Experiment x8A. Male rats treated with 4 mg/kg nephrilin peptide IM3N1 for 2 weeks in Example 5 were subsequently treated 3x weekly (Mon-Wed-Fri) for 4 weeks with vehicle (n=12) or with 1 mg/kg IM3v1p peptide (n=12) by subcutaneous bolus injection (200 uL volume). Longitudinal wound length between the hairlines was measured at 6 weeks, and this value was corrected for nose-to-ear length and width of each animal. The mean resulting value (in arbitrary units) is shown below. Wound length is reduced by one-third by treating with IM3v1p from weeks 2-6. * p<0.05, ** p<0.01 IM3v1p vs vehicle

TABLE-US-00016 Burn Group Weeks 2-6 Weeks 2-6 (n = 4) (vehicle) (1 mg/kg IM3vlp) Vehicle 127.1 ± 22.0 89.7 ± 17.7* Nephrilin IM3N1 131.3 ± 18.6 92.4 ± 19.5* Nephrilin IM3N1 + zinc 121.7 ± 24.8 83.2 ± 15.4* Nephrilin IM3N1 + iron 115.9 ± 5.7   72.6 ± 12.8**

Example 9. Iron and Calcium Stimulate the Binding of PI(4,5)P2 to Immodulator Peptides

[0083] Polyphosphoinositides (PPI) and in particular P1(4,5)P2, are among the most highly charged molecules in cell membranes, are important in many cellular signaling pathways.

[0084] Experiment x9A. Immodulator peptides IM1, 1M3 and 1M5 (400 ng/well) in PBS were bound to streptavidin-coated 96-well plates at room temperature for 60 minutes, in the presence or absence of 500 uM calcium chloride. Each well also received 200 ng cold PPI plus 10 ng FITC-labeled P1(4,5)P2 or P1(4)P1. The tables below show the relative fluorescence (with/without calcium background) expressed as a percentage of the highest binder, FITC-P1(4,5)P2+cold P1(3,5)P2, which was 2,496 fluorescence units. Background binding without calcium was, in all cases, 200-300 fluorescence units. The results show that 1M3 is the only one of the 3 peptides to bind P1(4,5)P2— but not (PI(4)P1— in a calcium-dependent manner. Excess PPI bisphosphates (but not triphosphate, or any of the monophosphates) dramatically stimulated binding, especially P1(3,5)P2. This synergy was as expected (see above).

TABLE-US-00017 IM1 IM3 IM5 cold PPI Mean SD P value Mean SD P value Mean SD P value FITC-PI(4,5)P) None 1.6 1.5 NS 2.2 2.7 NS 2.6 3.5 NS 3-P1 3.1 3.0 NS 1.7 0.2 NS 0.3 3.5 NS 4-P1 1.3 2.7 NS 6.3 3.2 NS 2.3 0.9 NS 5-P1 0.5 2.9 NS 10.4 2.9 NS 2.7 1.2 NS 3,4-P2 1.1 3.7 NS 51.5 7.6 0.0450 12.3 8.1 NS 4,5-P2 0.8 1.8 NS 63.7 7.7 0.0356 7.6 8.5 NS 3,5-P2 4.3 2.1 NS 100.0 1.0 0.0052 16.9 11.5 NS 3,4,5-P3 0.1 2.2 NS 3.2 0.5 NS 0.2 1.4 NS FITC-PI(4)P1 None 1.6 1.5 NS 2.5 2.9 NS 2.1 1.2 NS 3-P1 3.1 3.0 NS 2.5 0.3 NS 0.9 2.9 NS 4-P1 2.7 3.0 NS 2.4 0.7 NS 0.8 2.4 NS 5-P1 0.5 2.9 NS 1.9 1.4 NS 0.4 1.7 NS 3,4-P2 2.6 1.5 NS 2.4 0.6 NS 1.7 0.0 NS 4,5-P2 0.8 1.8 NS 0.1 0.5 NS 1.8 1.5 NS 3,5-P2 4.3 2.1 NS 5.6 0.9 NS 1.3 1.9 NS 3,4,5-P3 0.1 2.2 NS 1.2 0.5 NS 1.2 1.0 NS

[0085] The binding of PPIs by immodulator peptides (or the IGFBP proteins from which their sequences were derived) has never previously been shown.

[0086] Experiment x98. Binding of immodulator peptides to Echelon PPI Arrays. PPI arrays were purchased from Echelon Biosciences (Sal Lake City, UT) and used according to the manufacturer's recommendations. Colorimetric readout from each 100 pmol PPI spot in the array is expressed as 0=no binding, 1=low binding, 2=medium binding and 3=high binding. The results are shown below. The results show binding of 1M3 and 1M5 (and, to a lesser extent, 1M6) to P1(4,5)P2 but not phosphoinositol or P1(4)P1, except that 1M5 does show some binding to P1(4)P1. In addition, 1M3 and 1M5 show slight binding to P1(3,4,5)P3.

TABLE-US-00018 IM1 IM2 IM3 IM4 IM5 IM6 PI 0 0 0 0 0 0 PI(4)P1 0 0 0 0 2 0 PI(4,5)P2 0 0 3 0 3 1 PI(3,4,5)P3 0 0 1 0 1 0

[0087] Experiment x9C. NTA-coated 96-well plates were charged with either 500 uM zinc chloride or 500 uM ferric chloride and washed. Immodulator peptide 1M3 (100 ng/well) and FITC-P1(4,5)P2 (50 ng/well) were added, with or without 2 ug/well cold PPI, and incubated at room temperature for 60 minutes. The plate was washed and counted in a fluorometer. The results are shown below. The results show that NTA-bound iron can bind 1M3 peptide, and binding is enhanced by P1(3,5)P2 but not by P1(3)P1. *p<001 versus no metal control; #p<0.01 versus ferric iron alone.

TABLE-US-00019 Metal Cold PPI Mean ± SD No metal None 0.0 ± 0.2 Zinc None  2.2 ± 0.3*# Ferric iron None 80.1 ± 1.8* Ferric iron PI(3)P1 78.5 ± 5.8* Ferric iron PI(3,5)P2 100.0 ± 3.2*#

[0088] Experiment x9D. To a streptavidin-coated 96-well plate, 400 ng peptide 1M3 or 1M5 were bound per well. Each well received 100 uL of sodium acetate buffer pH5.2 containing 500 uM EDTA, Calcium Chloride, Zinc Chloride or Ferric

[0089] Chloride. Each well additionally contained 50 ng FITC-P1(4,5)P2 plus 2 ug of either cold P1(3,4)P2, P1(4,5)P2, P1(3,5)P2 or buffer. All assays were done in sextuplicate. After incubation at room temperature for 60 mins, wells were washed and counted in a fluorometer set at FITC excitation/emmission wavelengths. The results are shown below. At low pH conditions of this experiment, cold PPI did not enhance binding of immodulator peptide to P1(4,5)P2 as much in the presence of calcium, but did so dramatically in the presence of ferric iron.

TABLE-US-00020 IM3 peptide: EDTA Calcium Zinc Ferric Iron Buffer 30 ± 22 142 ± 29*  138 ± 388* .sup. 772 ± 168*# Cold PI(3,4)P2 26 ± 25 48 ± 61 65 ± 16 2,162 ± 269*# Cold PI(4,5)P2 10 ± 16 32 ± 23 21 ± 22 1,464 ± 320*# Cold PI(3,5)P2 63 ± 26 88 ± 22 118 ± 43  2,036 ± 303*# *p < 0.001 vs buffer control; #p < 0.001 vs ferric iron

TABLE-US-00021 IM5 peptide: EDTA Calcium Zinc Ferric Iron Buffer  88 ± 23  234 ± 34* 236 ± 55* 1,111 ± 167*# Cold PI(3,4)P2 150 ± 81 205 ± 50 151 ± 51  2,150 ± 420*# Cold PI(4,5)P2  81 ± 95 163 ± 80 104 ± 21  1,447 ± 304*# Cold PI(3,5)P2 205 ± 71  489 ± 146* 353 ± 29* 2,026 ± 176*# *p < 0.001 vs EDTA control; #p < 0.001 vs calcium and zinc

Example 10. Anti-Cancer Immodulator Peptides

[0090] Immune modulation functions of immodulator peptides have great potential untility in the field of cancer. A375 cell line was obtained from American Type Culture Collection (ATCC). They were grown in a T-75 flask in DMEM Medium containing 10% fetal bovine serum and penicillin-streptomycin at 37° C. in a humidified, 5% CO.sub.2 incubator. Cells (100 ml, 2,000 cells/well) were plated in a 96-well plate and incubated overnight at 37° C. in a humidified, 5% CO2 incubator. Next day, 10 ml/well of compounds were added (quadruplicate wells). After 72 hour incubation with the compound, cell viability was measured in a luminometer after the addition of 100 mL/well CellTiterGlo reagent (Promega Inc, Madison, Wis.) as recommended by the manufacturer.

[0091] Experiment x10A. Anti-cancer activity of immodulator peptides on A375 cells. A 96-well plate was seeded with 2,000 A375 cells per well in DMEM medium containing 10% FBS and PenStrep. After 24 hrs at 37 deg C, compounds and peptides were added (each treatment done in quadruplicate). After a further 72 hours incubation, 100 uL/well of CTG assay reagent purchased from Promega Inc. (Madison, Wis.) was added. Plate was read after 10 minutes, as recommended by the manufacturer. Peptides were added at 2 uM. The results of this experiment are shown below, expressed as percent survival of A375 cells. They show that anti-melanoma activity of various immodulator peptides are influenced by the core immodulator sequence, extension sequences, N-terminally conjugated carboxylic acids, and RIG-I co-inducer (4 uM). *p<0.05, **p<0.01 vs “no peptide” control; Kin1400=4 uM (RIG-I agonist); vlp=valproic; dec=decanoic; lau=lauric; rg108=RG108; h4p=h4-pentynoic; bpa=bromopyruvic; nd=not determined.

TABLE-US-00022 Peptide Sequence Buffer Kin1400 No peptide 100 100.7 IM1 KNGFYHSRQCETSMDGEAGLCW 102.1 nd IM2 KHGLYNLKQCKMSLNGQRGECW 99.0 nd IM3 KKGFYKKKQCRPSKGRKRGFCW 100.3 62.2** IM4 RNGNFHPKQCHPALDGQRGKCW 98.8 nd IM5 RKGFYKRKQCKPSRGRKRGICW 103.4 nd IM6 HRGFYRKRQCRSSQGQRRGPCW 102.2 Nd IM3avd KKGFYKKKQCRPSKGRKRGFCWAVD 96.0 12.5** IM3fvs KKGFYKKKQCRPSKGRKRGFCWFVS 96.1 22.6** IM3vlp (vlp)-KKGFYKKKQCRPSKGRKRGFCW 93.9** 32.6** IM3dec (dec)-KKGFYKKKQCRPSKGRKRGFCW 89.0** 53.8** IM3lau (lau)-KKGFYKKKQCRPSKGRKRGFCW 96.6* 17.4** IM3rg8 (rg108)-KKGFYKKKQCRPSKGRKRGFCW 89.9** 105.8 IM3h4p (h4p)-KKGFYKKKQCRPSKGRKRGFCW 91.7** Nd IM3K1 SLNPEWNETKGFYKKKQCRPSKGRKRGFCW 92.5** 17.0** IM3K1bpa (bpa)SLNPEWNETKGFYKKKQCRPSKGRKRGFCW 98.4 Nd IM3K1.11 SLNPEWNETKKGFYKKKQCRPSKGRKRGFCW 95.3* Nd IM2K1 SLNPEWNETHGLYNLKQCKMSLNGQRGECW 96.2 Nd

Example 11. Collagen Stimulating Activity of Immodulator Peptides

[0092] Immune modulation functions of immodulator peptides have potential untility in the field of cosmetics. HFF-1 cell line was obtained from American Type Culture Collection (ATCC). Cells were grown in a T-75 flask in DMEM Medium containing 10% fetal bovine serum and penicillin-streptomycin at 37° C. in a humidified, 5% CO.sub.2 incubator. Cells (100 ml, 2,000 cells/well) were plated in a 96-well plate and incubated overnight at 37° C. in a humidified, 5% CO2 incubator. Next day, 10 ml/well of compounds were added (quadruplicate wells). After 72 hour incubation with the compound, supernatants were collected for Collagen-1 ELISA assay and cell viability was measured in a luminometer after the addition of 100 mL/well CellTiterGlo reagent (Promega Inc, Madison, Wis.) as recommended by the manufacturer.

TABLE-US-00023 Peptide Sequence COL1 No peptide  100 IM1 KNGFYHSRQCETSMDGEAGLCW   98 IM2 KHGLYNLKQCKMSLNGQRGECW  109 IM3 KKGFYKKKQCRPSKGRKRGFCW  101 IM4 RNGNFHPKQCHPALDGQRGKCW  105 IM5 RKGFYKRKQCKPSRGRKRGICW   93 IM6 HRGFYRKRQCRSSQGQRRGPCW  112 IM3bex (bex)-KKGFYKKKQCRPSKGRKRGFCW  102 IM3isf (isf)-KKGFYKKKQCRPSKGRKRGFCW   131* IM3vlp (vlp)-KKGFYKKKQCRPSKGRKRGFCW    157** IM3dec (dec)-KKGFYKKKQCRPSKGRKRGFCW  114 IM3cin (cin)-KKGFYKKKQCRPSKGRKRGFCW  106 IM3rhn (rhn)-KKGFYKKKQCRPSKGRKRGFCW     68** IM3K9 AFNSYELGSKGFYKKKQCRPSKGRKRGFCW    155** IM3K9.1 AFNSYELGSKKGFYKKKQCRPSKGRKRGFCW    156** IM3K9c AFNSYELGSKGFYKKKQCRPSKGRKRGFCWAVDKY    158** IM3K8 FNSYELGSLKKGFYKKKQCRPSKGRKRGFCW   98

[0093] Experiment x11A. Collagen stimulating activity of immodulator peptides in HFF-1 dermal fibroblasts. Peptides were added to cells at 2 uM. COL1 immunoreactivity was measured in the supernatants of cultured cells by ELISA using a rabbit monoclonal anti-COL1 primary antibody (Abcam, Cambridge, Mass.). The results of this experiment are shown in Table above. Control (buffer) value of immunoreactivity was set to 100. The data show that collagen stimulating activity of various immodulator peptides are influenced by specific extension sequences and by N-terminally conjugated carboxylic acids. *p<0.05, **p<0.01 vs “no peptide” control; bex=bexarotene; isf=isoferulic; vlp=valproic; dec=decanoic; cin=cinnamic; rhn=rhein;

[0094] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.