GB2285047A - Polypeptides, rich in proline, which inhibit the NADPH oxidase system - Google Patents

Abstract

Polypeptides comprising the sequence PXXPPXP (wherein X is an amino acid), especially PAVPPRP or QPAVPPHPS, which inhibits at least one interaction between p47phox and p67phox of the NADPH oxidase system, and homologues thereof, are described. The polypeptides may be used in the therapy of inflammatory states.

Description

Polypeptides Which Inhibit The NADPH Oxidase Complex The present invention relates to polypeptides which inhibit at least one interaction between p47phox and p67Phox of the NADPH oxidase complex.

There is evidence to show that much of the tissue damage which occurs in inflammatory responses such as ischemia reperfusion injury and chronic rheumatic diseases is due to the production of free radicals such as O2. These free radicals are thought to be produced by the NADPH oxidase complex which includes the two proteins p47phox and p67Phox The p47phox interacts with at least one SH3 domain of p67Ph X. SH3 domains are thought to be involved in mediating protein-protein interactions between signalling components downstream of membrane bound receptors (Pawson, T. and Schlessinger, J. Current Biology 3, 434-442 (1993). Recently, a number of potential SH3 domain binding protections have been reported.The first of these, 3BP-1, binds to the SH3 domain of c-Abl in vitro and contains a region with sequence homology to the GTPase activating protein for the monomeric GTPase, Rho [Cicchetti, P. et al. Science 257, 803-807]. In addition, the signalling pathway from cell-surface receptors to Ras utilises an adaptor protein Grb2 which couples to activated receptors through an SH2 domain and interacts with a downstream signalling component, Sos, via at least one SH3 domain [Buday, L. and Downward, J. Cell 73, 611-620 (1993), Egam S.E. et al Nature 363, 45-51 (1993), Li, N. et al. Nature 363, 85-88 (1993), Olivier, J. P. et al. Cell 73, 179,-191 (1993), Rozakis-Adcock, M. et al Nature 363 83-85 (1993).

Previous reports have suggested that proline rich -sequences are important for the recognition of SH3 domains [Ren, R. et al, Science 259, 1157-1161 (1993), Buday and Downward 93, Egan 93, Le 93, Olivier 93, Rozakis-Adcock 93].

The SH3 binding motif of 3BP1 has been previously identified as APTMPPPLPP with the proline residues at positions 2, 7 and 10 appearing to be crucial for binding to the c-Abl SH3 domain.

Similarly, the corresponding sequence of a second SH3 domain binding protein, 3BP2, has been identified as PAYPPPPVP and the consensus sequence XPXXPPPZXP (where X is any amino acid and Z is hydrophobic) has been proposed.

The present invention provides synthetic polypeptides which competitively inhibit the binding of p47phoX and p67PhoX; p47Phox and p67PhOX both have two SH3 domains; we have found that the carboxy-terminal SH3 domain of p67Phox interacts with a proline rich region near the carboxy terminal of p47phox.

According to the present invention there are provided polypeptides which comprise 8 or 9 amino acids having the respective sequences QPAVPPRP and QPAVPPRPS and which inhibit at least one interaction between p47phoX and p67Phox These sequences are significantly different from the above prior consensus sequences. Homologues of the sequences QPAVPPRP and QPAVPPRPS and homologues of extended forms of these sequences are also provided by the present invention.

The present invention also provides polypeptides which show substantial homology to the proline rich region near the carboxy-terminal of p47phoX. They bind to the carboxy-terminal SH3 domain of p67Phox and inhibit at least one intereaction between p67phox and p47phox Also provided by the present invention are polypeptides which comprise the following amino acid sequences QPAVPPRPS; QPAVPPHPS; QPAVPPHP; CPAVPPRPS; CPAVPPRP; NPAVPPRPS; NPAVPPRP; GPAVPPRPS; GPAVPPRP; SPAVPPRPS; SPAVPPRP; TPAVPPRPS; TPAVPPRP; YPAVPPRPS;YPAVPPRP; KPQPAVPPRPSADL; and BKPQPAVPPRPSADL (where B is a myristyl or palmytoyl group) and which inhibit at least one reaction between p47Phox and p67PhoX. It can be seen that some of the amino acid sequences shown above are homologues of the sequences QPAVPPRPS and QPAVPPRP.

Also provided by the present invention are polypeptides which show substantial homology to the above polypeptides.

Prom experimental evidence it appears that it is preferable to have proline residues at positions n, n+3, n+4, n+6 (where n is a positive integer) with respect to each other; thus the present invention also provides polypeptides having proline at positions n, n+3, n+4, n+6 (where n is a positive integer) with respect to each other.

The present invention is illustrated by the following experimental work: Affinity matrices were prepared to establish the role of the SH3 domains by immobilising various glutathione Stransferase (GST)-SH3 domain fusion proteins on glutathione Sepharose beads. These matrices were used to purify proteins from DMSO-differentiated HL60 cell extracts which specifically bound to the SH3 domains. A major SH3-binding protein was a polypeptide of 47 kDa which specifically bound to the Cterminal SH3 domain of p67Phox (p67PhoxC) but not to any of the other SH3 domains tested. This protein was subsequently identified as p47phoX by protein purification and sequence analysis.

Analysis of the p47Pbox sequence revealed the presence of the proline rich motif near the C-terminus of the protein.

To define the binding site for p67Phcx, a synthetic peptide corresponding to this proline rich region of p47phox was used in competition experiments. A synthetic peptide with the sequence KPQPAVPPRPSADL (Peptide P2), corresponding to the p47phox proline rich motif, inhibited the binding of the carboxyterminals of p47Pbox and p67Phox to each other at a concentration of 750pM. Peptides corresponding to proline rich sequences in p67Ph X, cytochrome b245, PTPase 1B and dynamin all failed to compete at the same concentration although of these proteins only dynamin has been shown to bind SH3 domains in vitro [Gout, I. et al. Cell 75, 25-36 (1993).Dose response studies showed an IC50 for binding inhibition of about 100M.

A series of truncated variants of the synthetic peptide P2 were tested in the binding assay to further characterise the putatative SH3 domain binding site. The minimum peptide sequence tested which competed for the binding of p47phoX to p67Phox was QPAVPPRPS. The binding site for p67Phox, although proline rich, does not match the consensus sequence of the prior art.

The invention is further illustrated with reference to Figures 1 to 5 of the accompanying drawings Figure 1 shows the results of in vitro binding of P47phox to the C-terminal SH3 of p67Phox followed by amino acid sequence analysis of the bound protein. The amino acid sequence of p47phoX is shown in the upper sequence and the derived sequences from 3 peptides from the 47 kD protein which binds to p67Phox SH3 [Pep 1, Pep 2, Pep 3]. The derived peptide sequences are 100% homologues with regions of p47phoX.

GST-SH3 fusion proteins were expressed in bacteria and purified as described previously, D.B. Smith and Johnson, K.S.

Gene 67, 31-40 (1988). Affinity matrices were prepared by immobilising the fusion protein glutathione beads. HL60 cells, differentiated for 5 days in 1.25% (v/v) DMSO were solubilised in lysis buffer (50mM Tris pH 7.5, 5 mM EGTA, 2W (v/v) Triton X-100 (TM), 75 Mm NaCl, o.5 Mm PMSF). After centrifugation at 14,000g for 15 minutes, the supernatant containing solubilised protein was incubated with the SH3 domain affinity resins for two hours 4"C. The beads were then washed extensively in wash buffer (50 mM Tris pH 7.5, 0.1 k (v/v) Triton X-100 (TM), 10% (v/v) glycerol) and resuspended in SDS-PAGE sample buffer.

After boiling, proteins were separated by SDS-PAGE, detected by Coomassie blue staining and p47 was excised and digested with trypsin, peptides were extracted from the gel and separated by ion exchange and reverse-phase hplc. Purified peptides were sequenced using fast cycle, automated Edman chemistry on an Applied Biosystems 477A (Totty et al Prot.Sci.1, 1215-1224 (1992).

Amino acid sequences of six proline rich peptides were synthesised: Peptide P1, dynamin; Peptide P2, p47phoX; Peptide P3, p67PhOX; Peptide P4, cytochrome B245; Peptide P5, PTPase 1B; P6 vinculin. Affinity matrices and HL60 cell extracts were prepared as described for Fig 1. Solubilised protein was incubated with the p67PhoxC SH3 domain affinity resin in the presence of the proline rich polypeptides at a concentration of 750cm. The beads were washed and SDS-PAGE samples were prepared as described for Fig 2.

Figure 2 shows results of an inhibition assay of p47Phox - p67PhOX binding by proline rich peptide, P2. In vitro binding of p47Pbox to the C-terminal SH3 domain of p67Phox was assayed in the presence of proline rich synthetic peptides derived from dynamin (track 4), p47phox (track 5), p67Phox (track 6), cytochrome B245 (track 7), PTPase 1B (track 8), vinculin (track 9) and in the absence of any peptide (track 3). Figure 2 is a silver stained gel and Table 1 summarises the binding data.

Figure 3 shows localisation of the p67Ph X binding site on p47phox.. HL60 cell extracts were incubated with p67Pbox affinity matrices in the presence of a proline rich peptide derived from p47phoX (P2) and P2 peptides truncated at the N- or C- terminus or at both. Silver stained gel is shown.

TABLE 2 lists the polypeptides whose binding results are shown in Figure 3.

Binding of p47phoX to immobilised p67Phox was assayed as described previously. Solubilised protein was incubated with the p67Phox carboxy-terminal SH3 domain affinity resin in the presence of the proline rich peptides at a concentration of 400pom. Bound proteins were separated by SDS-PAGE and visualised by silver staining.

Figure 4 is a schematic of the process involved for identification of SH3 binding proteins. This process is carried out by isolating the SH3 domain and adding a suspected binding protein. Washing is followed by running the protein mixture on an SDS gel. Bound and unbound protein are then identified.

Figure 5 is a schematic of interactions involved in the NADPH oxidase complex.

Polypeptides according to the invention and equivalent derivatives thereof, and medicaments containing them together with conventional pharmaceutical carrier or excipient, can be used for the treatment of chronic and acute inflammatory diseases, and conditions, including but not limited to septic shock, rheumatoid and other arthritides, asthma, adult respiratory distress syndrome and other pulmonary inflammatory disorders, ischaemic heart disease, reperfusion injury and inflammatory bowel disease! TABLE Inhibition or p47 binding to p67c-@@@ domain by proline-rich peptides Peptide Protcin Sequon@e Inhibition of No. p 47: :p67C-SH3 binding P1 Dynamin PAVPPARPRGSGPAPGPPPAG P2 P47@@@ XPQPAVPPRPSADL ++ P3 P67@@@ APLQPQAAEPPPRPKTPE P4 Cyt. B245α KQPPSNPPPRPPAEA P5 PTP@@@ 1B DLEPPPEHIPPPPRPPKR +/ P6 Vinculin APPKPPLPEGEVPPPRPPPPE Table 2 PEPTIDE INHIBITORY ACTIVITY DPQPAYPPRPSADL + PQPAVPPRPSADL + QPAVPPRPADL + PAVPPRPsADL AVPPRPSADL + VPPRPSADL PPRPBADL + PRPBADL + RPSADL KPQPAVPPRPSAD + DPQPAVPPRPSA + PRQPAVPPRPS + QPAVPPRPS + QPAVPPRP + QPAVPPR Pharmacore QPAVPPRP

Claims (7)

1. CLAIMS 1. A polypeptide which consists of or includes 8 amino acids having the sequence QPAVPPRP and which inhibits at least one interaction between p47PX and p67phox of the NADPH oxidase system.
2. 2. A polypeptide which consists of or includes amino acids having any one of the following sequences QPAVPPRP QPAVPPRPS QPAVPPHPS CPAVPPRPS NPAVPPRPS GPAVPPRPS SPAVPPRPS TPAVPPRPS YPAVPPRPS KPQPAVPPRPSADL BKPQPAVPPRPSADL (where B is a myristyl or palmytoyl group) and which inhibits at least one reaction between p47phoX and p67Ph X.
3. 3. A polypeptide having substantial homology to a polypeptide according to claim 1 or 2.
4. 4. A polypeptide having proline at positions n, n+3, n+4, n+6 (where n is a positive integer) with respect to each other in an amino acid sequence and which inhibits at least one interaction between p47phoX and p67Phox of the NADPH oxidase complex.
5. 5. The use of polypeptide according to any preceding claims for the preparation of a medicament for the treatment of inflammatory disease or condition.
6. 6. A pharmaceutical composition comprising compound according to any of claims 1 to 4 with pharmaceutical carrier or excipient.
7. 7. A method of treating inflammatory disease or condition in a patient which comprises administering to the patient compound according to any of claims 1 to 4.