United States Patent: 6,849,596
Issued: February 1, 2005
Inventors: Holms; Rupert Donald (66 Regents Park Rd., London NW1 7SX, GB)
Appl. No.: 856070
Filed: May 17, 2001
PCT Filed: September 15, 2000
PCT NO: PCT/GB00/03566
371 Date: May 17, 2001
102(e) Date: May 17, 2001
PCT PUB.NO.: WO01/25275
PCT PUB. Date: April 12, 2001
This invention describes novel charged molecules which specifically bind to the Hepreceptor, a regulatory site which I have discovered in human ezrin. My invention is that when peptides or other charged molecules bind to the Hepreceptor, medically useful immune responses are induced. These charged molecules can be administered orally and by other routes for the treatment of various infectious diseases and cancer. I have determined that the Hepreceptor (human ezrin 308-373) comprises of two adjacent alpha helical domains which are folded together at a hinge region (M339-M340) and stabilised by complimentary side chain charges of the primary amino acid sequence in the soluble cytoplasmic conformation of ezrin. I have determined that in the unfolded membrane associated conformation of ezrin, the Hepreceptor is pushed through the cell membrane and is exposed on the outer surface of the cell. Hepreceptor-Domain A (amino acid numbers 308-339 of human ezrin), comprises of the following 32 amino acid sequence. SEQ ID 1 AREEKHQKQLERQQLETEKKRRETVEREKEQM Hepreceptor-Domain B (amino acid numbers 340-373 of human ezrin), comprises of the following 34 amino acid sequence (Tyrosine 353 [Y] may be phosphorylated to phosphotyrosine [Yp] in the membrane associated conformation of ezrin): SEQ ID 2 MREKEELMLRLQDY(p)EEKTKKAERELIEQIQRALQ.
Description of the Invention
BACKGROUND TO THE INVENTION
The field of the present invention relates to the treatment of infectious disease and cancer by inducing disease fighting immune responses. The growing problem of new strains of pathogenic bacteria resistant to antibiotics, the limited range of compounds effective against chronic viral and fungal infections and shortage of effective anti-cancer treatments demonstrates the need for compounds that can enhance the host defence against these medical problems. This invention relates to novel charged molecules which stimulate immune responses by binding to the Hepreceptor, a novel active site in human ezrin which I have discovered. The preferred charged molecules are novel peptides with sequences identical to the Hepreceptor in human ezrin.
Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins which play structural and regulatory roles in a wide range of cell types. There is considerable evidence to indicate that ezrin regulates the structure of the cortical cytoskeleton to control cell surface topography. Ezrin adopts two main conformations: 1) a soluble folded form which is found in the cytoplasm and. 2) an unfolded and elongated form which is found attached to the cytoplasmic surface of the cell membrane particularly in conduction with microvilli and other activation related structures. The N terminal domain of the protein is attached to the cytoplasmic surface of the membrane while the C terminal part binds to the actin cytoskeleton. Ezrin is a tyrosine kinase substrate in T cells and is also tyrosine phosphorylated as a result of Epidermal Growth Factor (EGF) stimulation of the EGF receptor. The N terminal domain of ezrin in its extended conformation binds to the cytoplasmic tail of CD44 in the presence of PIP2. Ezrin also may bind to the cytoplasmic tail of ICAM-2. Ezrin is very sensitive to regulatory proteases such as calpain and is rapidly turned over during cell activation.
Anthony Bretscher, David Reczek and Mark Berryman (1997).
"Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures" Journal of Cell Science 110: 3011-3018.
Detailed analysis of the secondary structure of ezrin shows that there are three main structural domains: an N terminal domain from amino acids 1 to 300, a highly charged alpha domain from amino acids 300 to 470 and C terminal domain from amino acids 470 to 585. Structural modelling suggests that the alpha domain is folded into two anti-parallel helices in the soluble globular form of ezrin although the location of the hinge has not been identified. In the model of the extended phosphorylated form, ezrin is attached to the inner surface of the cell membrane by the N terminal domain, the alpha domains of two ezrin molecules are paired into anti-parallel dimers and located below the cell surface membrane. In this extended form, ezrin is tyrosine phosphorylated at tyrosine 353 (Yp 353).
Ossi Turunen, Markku Sainio, Juha Jaaskelainen. Olli Carpen, Antti Vaheri (1998) "Structure-Function relationships in the ezrin family and the effect of rumor-associated point mutations in neurofibromatosis 2 protein" Biochimica el Biophysica Acta 1387: 1-16.
I disclosed in U.S. Pat. No. 5,773,573 that the fourteen amino acid peptide HEP1, (amino acid sequence of TEKKRRETEREKE, SEQ ID 28, identical to amino acids 324 -337 of human ezrin) which has a 70% identity to the C terminus of gp120 could inhibit HIV replication in vivo in man. At the time I believed that the observed anti-HIV effect of peptide HEP1 was due to the orally administered HEP1 inducing
TABLE 1 Amino acids, three letter code, one letter code and side chain charges CHARGES ON AMINO ACID SIDE CHAINS AT PHYSIOLOGICAL pH Three One letter letter Amino acid code code Charge Symbol Glycine Gly G NONE Alanine Ala A NONE Valine Val V NONE Isoleucine Ile I NONE Leucine Leu L NONE Serine Ser S NONE Threonine Thr T NONE Aspartic acid Asp D NEGATIVE -- Glutamic acid Glu E NEGATIVE -- Phosphotyrosine Tyr(P) Yp NEGATIVE -- Asparagine Asn N WEAK NEGATIVE - Glutamine Gln Q WEAK NEGATIVE - Lysine Lys K POSITIVE + Arginine Arg R POSITIVE + Histidine His H WEAK POSITIVE + Proline Pro P NONE Tryptophan Trp W NONE Phenylalanine Phe F NONE Tyrosine Tyr Y NONE Methionine Met M NONE Cysteine Cys C NONE
DETAILED DESCRIPTION OF THE INVENTION
The soluble conformation of ezrin found in the cytoplasm comprises of two adjacent alpha helical domains which are folded together at a hinge region (M339-M340) into two anti-parallel helices stabilised by complimentary side chain charges of the primary amino acid sequence. It is the subject of this invention that the positively and negatively charged side chains of the amino acid sequence of Hepreceptor Domain A are complementary to the positively and negatively charged side chains of the amino acid sequence of the Hepreceptor Domain B. In the activated open conformation of ezrin, the interaction of the Domain A and Domain B of the Hepreceptors of two different ezrin molecules allows the formation of anti-parallel dimers. In addition to the antiparallel dimers of ezrin which form below the cell surface, I have determined that these dimers can form between a Hepreceptor exposed on the surface on one cell with a Hepreceptor exposed on the surface of another cell. When the two Hepreceptors make contact during close association of two cell surfaces an activation signal is initiated in both cells (FIG. 2). Any charged molecule that partially mimics the interaction between the side chains charges of Domain A and Domain B of the Hepreceptors will give rise to a medically useful biological response.
Hepreceptor-Domain A (amino acid numbers 308-339 of human ezrin), comprises of the following 32 amino acid sequence.
(The sequence are listed using the single letter code for ch amino acid written from the N terminus to the C terminus of the polypeptide Yp represents the form of phosphotyrosine found in vivo).
SEQ ID 1 AREEKHQKQLERQQLETEKKRRETVEREKEQM
In U.S. Pat. No. 5,773,573, I disclosed the anti-HIV activity of peptide HEP1 (SEQ ID 28) which I have now discovered has a sequence identical to part of Hepreceptor-Domain A (spanning amino acids 324-337 of the human ezrin sequence). In the above patent I made the assumption that anti-HIV activity was due to the induction of immunological tolerance to autoreactive immune responses induced by the C terminus of HIV gp120. I can now disclose that the anti-HIV activity of HEP1 is due to its binding to Hepreceptor Domain B and the induction of a novel immune response. It is a subject of this invention that there are novel peptides derived from the Hepreceptor of ezrin with significantly superior activity to HEP1.
Hepreceptor-Domain B (amino acid numbers 340-373 of human ezrin), comprises of the following 34 amino acid sequence (Tyrosine 353 [Y] may be phosphorylated to phosphotyrosine [Yp] in the membrane associated conformation of ezrin):
SEQ ID 2 MREKEELMLRLQDY(p)EEKTKKAERELSEQIQRALQ
I have determined that Domain B of the Hepreceptor is the site on ezrin to which HIV gp120 binds during infection of the brain. (HIV gp120 binds to Hepreceptor Domain B using its charged C terminal amino acids which have a 70% identity to part of Hepreceptor Domain A). Novel charged molecules which bind to the Hepreceptor may be useful in treating HIV related dementia.
Claudia Hecker, Christoph Weise, Jurgen Schneider-Schaulies, Harvey Holmes, Volker ter Meulen (1997) "Specific binding of HIV-1 envelope protein gp120 to the structural membrane proteins ezrin and moesin." Virus Research 49 215-223.
I have demonstrated (EXAMPLE 1A) that Hepreceptor peptides have significant adjuvant activity and this is demonstrated by enhancing the IgG antibody response to Ovalbumin in mice using HEP1, Rupe312, Rupe1014. Rupe1024 and Rupe2032. I have also demonstrated (EXAMPLE 1B) the activity of Hepreceptor peptides in enhancing the antibody-dependent cytotoxic response in Thymus to Sheep Red Blood Cells (SRBC) in mice using HEP1. Rupe312, Rupe15, Rupe1014, Rupe1024 and Rupe2032.
(Rupe312 SEQ ID 8:KKRRETVERE and Rupe15 SEQ ID 3, TEKKR and Rupe1014 SEQ ID 16: EREKE and Rupe1024 SEQ ID 17: EREKEQMMREKEEL and Rupe2032 SEQ ID 19: KEELMLRLQDYEE and HEP1 SEQ ID 28: TEKKRRETVEREKE).
I have demonstrated (EXAMPLE 2) that hepreceptor peptides have significant anti-tumour activity and this is demonstrated by Hepreceptor peptides reducing the growth rate of fast growing transplanted sarcomas and slower growing transplanted cervical cancer in mice using Rupe312 and Rupe414.
(Rupe312 SEQ ID 8:KKRRETVERE and Rupe414 SEQ ID 13: KRRETVEREKE).
I have demonstrated (EXAMPLE 3) that HEP1 therapy (10 mg per day orally either for thirty days or ninety days) in 21 HIV infected patients induces immune responses which leads to clinical improvement over the following six months after therapy, as measured by an increasing CD4 T lymphocyte population and declining opportunistic infections, declining HIV infectivity and declining CD38.CD8 population of T lymphocytes (an established prognostic marker of the progression to AIDS).
M Levancher, F Hulstaert, S. Tallet, S Ullery, J J Pocidalo, B A Bach (1992).
"The significance of activation markers on CD8 lymphocytes in human immunodeficiency syndrome: staging and prognostic value" Clinical Experimental Immunology 90 376-382.
A mean increase in the level of expression of CD44 and MHC Class I on T lymphocytes over six months was observed which appears to also correlate with the clinical improvement. No toxicity was detected with the administration of HEP1. Increases of MHC Class I expression and CD44 expression are associated with increases in memory T cells and Class I restricted cell mediated immunity.
Stephan Oehen and Karin Brduscha-Riem (1998).
"Differentiation of Naive CTL to Effector and Memory CTL: Correlation of Effector Function with Phenotype and Cell Division" The Journal of Immunology 161 5338-5346.
The results of this trial demonstrates that a peptide or other charged molecule which mimics all or part of the Hepreceptor can give rise to an activation signal that eventually leads to a change in the homoeostasis of the immune system and long term up regulation of cell mediated and humoral immunity. I have also demonstrated that acute and chronic candida infection in women can be treated and cured by the immune response arising from Hepreceptor stimulation (EXAMPLE 4). I have demonstrated that peptides derived from the Hepreceptor can activate monocytes and macrophages in mice both in vitro and in vivo, which leads to a protective immune response. (EXAMPLE 5). Peptides of this invention have a significantly higher activity than HEP1. Hepreceptor stimulation also leads to the activation of human peripheral blood mononuclear cells which was demonstrated by measuring the incorporation of radioactive tritiated thymidine into DNA of the growing cells. Novel peptides, Rupe312 and Rupe414 derived form Hepreceptor Domain A had a ten fold higher activity than HEP1. (EXAMPLE 6).
(Rupe312 SEQ ID 8:KKRRETVERE and Rupe414 SEQ ID 13: KRRETVEREKE).
I also discovered that a 24 hour incubation of human White Blood Corpuscles (WBC) with peptides derived from the Hepreceptor results in a fall in MHC Class I cell surface expression, probably due to cell activation and receptor internalisation, and an increase in the total population of macrophages expressing MHC Class I. This is consistent with the long term increase in the population cells expressing MHC Class I seen in HIV patients during the six months following HEP1 therapy. In this assay system Rupe312 and Rupe414 had significantly higher activity than HEP1 (EXAMPLE 7).
(Rupe312 SEQ ID 8:KKRRETVERE and Rupe414 SEQ ID 13: KRRETVEREKE).
I have demonstrated (EXAMPLE 8) that hepreceptor peptides have significant suppressive effect on the expression of IL-8. The inhibition of IL-8 may play a role in the selective activity of hepreceptor peptides in activating monocytes/macrophages (Rupe312 SEQ ID 8:KKRRETVERE).
I have demonstrated (EXAMPLE 9) that very low dose Hepreceptor peptides (1-100 nanograms/mouse) protect mice from acute infection by Salmonella tryphimurium (Rupe15 SEQ ID 3: TEKKR and Rupe1024 SEQ ID 17: EREKEQMMREKEEL and HEP1 SEQ ID 28: TEKKRRETVEREKE).
I have demonstrated (EXAMPLE 10) that very low dose Hepreceptor peptides (1-1000 nanograms/mouse) enhance survival time in a mouse lethal herpes virus infection model. (HEP1 SEQ ID 28: TEKKRRETVEREKE).
This invention describes charged molecules which specifically bind to the Hepreceptor. I have designed three groups of novel charged peptides which have sequences identical to the amino acid sequences of the complementary domains of the Hepreceptor and which either bind to Hepreceptor Domain B (SEQ ID 3-SEQ ID 16), or to both Domain A and B (SEQ ID 17), or which bind to Domain A (SEQ ID 18-SEQ ID 27). The peptides which are a subject of this invention probably bind to cell surface exposed Hepreceptors and stabilise the unfolded conformation of ezrin and induce immuno-modulatory effects. The preferred peptides are between five and thirteen amino acids in length and the preferred sequences are as follows.
Hepreceptor Domain B binding peptides:
SEQ ID 3
SEQ ID 4
SEQ ID 5
SEQ ID 6
SEQ ID 7
SEQ ID 8
SEQ ID 9
SEQ ID 10
SEQ ID 11
SEQ ID 12
SEQ ID 13
SEQ ID 14
SEQ ID 15
SEQ ID 16
Hepreceptor Domain A and Domain B binding peptide:
SEQ ID 17
Hepreceptor Domain A binding peptide:
SEQ ID 18
SEQ ID 19
SEQ ID 20
SEQ ID 21
SEQ ID 22
SEQ ID 23
SEQ ID 24
SEQ ID 25
SEQ ID 26
SEQ ID 27
Other peptides or other charged molecules which bind to Domain A or Domain B or bridge Domain A and Domain B of the Hepreceptor are likely to be biologically active. These peptides or other charged molecules can be administered orally and by other routes for the treatment of various infectious diseases and cancer.
How to Make
Peptides used in this invention may be synthesised for example, using a solid phase method using either Boc or Fmoc chemistry or any other practical route for peptide synthesis known to those skilled in the art of peptide synthesis.
Stepwise solid phase synthesis with Boc-amino acids can be performed based on the method of Merrifield; (Journal of American Chemical Society 85 2149-2154). The following compounds can be used (Novabiochem resin: Boc-Glu(OBzl)-PAM, Amino acids: Boc-Lys (2Cl--Z--)OH, Boc-Glu(OBzl)OH, Boc-Arg(Tos)OH, Boc-Val-OH. Boc-Thr (Bzl)--OH, Solvents: DMF (Rathburn), Dichloromethane (BDH), Ethylacetate (BDH). Reagents: HBTU (Phase Separations Ltd), p-Cresol (Lanchester) TFA (Halocarbon Products Corporation) HF (BOC) DIEA (Fluka). Recommended reactive side chain protecting groups for Boc-amino acids are:, Arg (Tos), Asn (Xan), Asp (OcHxl), Glu (OcHxl) Gln (Xan) or Gln, His (DNP), Lys (CIZ). Serine (Bzl) Tyr (BrZ) Trp (CHO). The abbreviations have the following meanings: DCC=Dicyclohexylcarbodiimide. DIC=Diisopropylcarbodiimide. DCM=Dichloromethane, DMF=Dimethylformamide, TFA=Trifluoracetic acid, Boc=t-Butyloxycarbonyl, HOBT=Hydroxybenzotriazole, DIEA=Diisopropylethylamine, DCU=Dicyclohexylurea.
For example, boc synthesis of a peptide of this invention could be performed as follows: HBTU activation/in situ neutralization on 0.5 mmol scale uses 0.5 mmol of resin and a three fold excess of activated Boc amino acid. Boc amino acid and activating reagent (HBTU) should be used in equimolar quantities ie 2 mmol each in this case equals a 3xexcess. DIEA is used to both neutralise the resin for coupling and to activate the Boc-amino acid. (Hence 2.5 mmol is used. 1 equivalent Boc-aa and 1 equivalent resin). Reagents: 0.5M HBTU in DMF (MW=379, 0.5M=18.95 g in 100 ml, note it is not light stable) requires 2 mmol=4 ml and 2.5 mmol DIEA=0.46 ml (MW=129, d=0.742). Activation of aminoacids: Boc-amino acid should be activated only immediately prior to addition to the resin, especially in the case of Arg (Tos). For all Boc amino acids: weigh 2 mmol Boc amino acid into a 20 ml glass sample bottle. Add 4 ml 10.5M HBTU solution and shake to dissolve solid. Add 0.46 ml DIEA and mix (some colour change may be observed). Method: wash resin with DMF, remove Boc-protecting group with 100% TFA--Shake twice for 1 minute draining in between, drain, flow wash with DMF for 1 minute, drain, add activated amino acid solution, shake for 10 minutes, then take sample and perform the ninhydrin test to determin coupling efficiency. On completion of the synthesis flow wash with DMF, then DCM and dry. The synthesis of the first and every subsequent level of peptide construction is achieved using a three fold excess of HBTU activated Boc-amino acids in DMF. In all couplings, the coupling efficiency should be more than 99% as indicated by quantative ninhydrin testing. Deprotection of the N-termini is performed in 100% TFA. The resin peptide is carefully flow-washed before and after the deprotection. After the last coupling and removal of the Boc-protection, the peptide resin is washed with dichloromethane and dried by air. The peptide is removed from the resin support by the high HF method (2 g resin peptide, 2 g cresol, 20 ml HF, 1.5 h at -5oC.) to yield the crude peptide which is precipitated with ethylacetate (100 ml) and redissolved in 6M guanidine HCL-0.1M TRIS solution (20 ml).
The peptide can be purified as follows using an analytical HPLC separation on a Vydac C18 5 RAC column. HPLC grade acetonitrile (aldrich) and water is filtered through a membrane filter and degassed with helium flow prior to use. Analytical separation achieved with a solvent gradient beginning with 0% acetonitrile, increasing constantly to 60% acetonitrile at 20 minutes, staying at this concentration for twenty minutes and decreasing steadily to 0% acetonitrile for 10 minutes at a constant flow of 1.2 ml per minute. Preparative separation of peptide achieved on a TSK-GEL preparative C18 column. Separation is achieved with a solvent gradient beginning with 0% acetonitrile, increasing constantly to 18% acetonitrile at 60 minutes, then 60% acetonitrile for 80 minutes, staying at this concentration for 30 minutes at a constant flow of 8 ml per minute. The gradient can be controlled by two microprocessor controlled Gilson 302 single piston pumps. Compounds can be detected with a Waters 486 Tunable Absorbance Detector at 214 nm and analytical chromatographs recorded with an HP laserjet 5L. A Holochrome UV-VIS detector 220 nm for preparative chromatographs can be recorded with an LKB 2210 single channel recorder. Capillary Electorphoresis quality control can be carried out using Waters Quanta 4000 equipment using a phosphate buffer (75 microM) pH2.5 run at 15 kV, sample time 20 seconds, loaded by hydostatic injection on 60 cm column, run time 12 minutes. The yield for 1 g 0.46 mmol resin synthesis should be about 250 mg pure peptide
Alternative solution synthesis methods may also be used to produce larger quantities of the peptides of this invention. Protected trimer fragments can be obtained using stepwise synthesis by the active esters method known to those skilled in the art of peptide synthesis. The fragments are then assembled using DCC/HOBT after removal of relevant C and N terminal protective groups. After removal of all protective groups the crude peptide is partially purified on SP-Sephadex-C25 ion exchange chromatography followed by preparative HPLC then lyophilised.
How to Use
0.01 to 1000 mg of lyophilised peptide may be dissolved in 1-10 ml distilled water and administered orally or vaginally. 0.01 to 1000 mg may be formulated in to a pill or capsule or suppository with carriers used commonly by those skilled in the art of pill or capsule or suppository manufacture and administered orally or vaginally or anally. A filter sterilized solution of between 0.001 and 100 milligrams of peptide in distilled water may be injected subcutaneously or intravenously or intramuscularly.
Claim 1 of 20 Claims
What is claimed is:
1. An isolated molecule that binds to the hepreceptor, wherein said molecule consists of 5 to 41 amino acids containing at least 5 consecutive amino acids of SEQ D NO:29.