Cell-penetrating compstatin analogs and uses thereof
In some aspects, the present invention cell-penetrating compstatin analog and compositions comprising cell-penetrating compstatin analog. In some aspects, the invention further provides methods of using cell-penetrating compstatin analogs treat a complement-mediated disorder, e.g., to inhibit complement-mediated damage to a cell, tissue, or organ, to inhibit production or release of biologically active C3 cleavage products.
1. A method of characterizing a cell-penetrating compstatin analog (CPCA), the method comprising: (a) contacting a cell that expresses primate C3 with a CPCA in vitro; and (b) assessing the activity of the CPCA, wherein assessing the activity of the CPCA comprises exposing the cell to a stimulus that would, in the absence of a CPCA, cause release of C3 cleavage products from the cell; and measuring the amount of one or more C3 cleavage products released from the cell, and wherein the CPCA comprises:
(i) a compstatin analog moiety comprising a peptide comprising an amino acid sequence of one of SEQ ID NOs: 3-6 and 9-36; and
(ii) a cell penetrating moiety (CPM) comprising a cell penetrating peptide comprising an amino acid sequence of one of SEQ ID NOs: 77-136,
wherein the compstatin analog moiety is covalently linked to the CPM.
2. The method of claim 1 , wherein the compstatin analog moiety is linked to the CPM via a linker.
3. The method of claim 1 , wherein the compstatin analog moiety comprises a cyclic peptide having a core sequence of X′aa-Gln-Asp-Xaa-Gly (SEQ ID NO: 3), where X′aa and Xaa are selected from Trp and analogs of Trp.
4. The method of claim 1 , wherein the compstatin analog moiety comprises a cyclic peptide having a core sequence of X′aa-Gln-Asp-Xaa-Gly-X″aa (SEQ ID NO: 4), where X′aa and Xaa are each independently selected from Trp and analogs of Trp, and X″aa is selected from His, Ala, single methyl unbranched amino acids, Phe, Trp, and analogs of Trp.
5. The method of claim 1 , wherein the compstatin analog moiety is a compound that comprises a cyclic peptide having a sequence of X′aa1-X′aa2-X′aa3-X′aa4-Gln-Asp-Xaa-Gly-X″aa1-X″aa2-X″aa3-X″aa4-X″aa5 (SEQ ID NO: 5), where X′aa4 and Xaa are selected from Trp and analogs of Trp, wherein X′aa1, X′aa2, X′aa3, X″aa1, X″aa2, X″aa3, X″aa4, and X″aa5, are independently selected from among amino acids and amino acid analogs, wherein the peptide is cyclized via a bond between X′aa2 and X″aa4.
6. The method of claim 1 , wherein the compstatin analog moiety is a compound that comprises a cyclic peptide having a sequence:
Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa2*-Gly-Xaa3-His-Arg-Cys-Xaa4 (SEQ ID NO: 6); wherein:
Xaa1 is Ile, Val, Leu, B 1 -Ile, B 1 -Val, B 1 -Leu or a dipeptide comprising Gly-Ile or B 1 -Gly-Ile, and B 1 represents a first blocking moiety, wherein B 1 comprises an acetyl group;
Xaa2 and Xaa2* are independently selected from Trp and analogs of Trp;
Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;
Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Ala and Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn optionally is replaced by a second blocking moiety B 2 wherein B 2 comprises an —NH 2 group; and
the two Cys residues are joined by a disulfide bond.
7. The method of claim 6 , wherein
Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprising Gly-Ile or Ac-Gly-Ile;
Xaa2 and Xaa2* are independently selected from Trp and analogs of Trp;
Xaa3 is His, Ala or an analog of Ala, Phe, Trp, or an analog of Trp;
Xaa4 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide selected from Thr-Ala and Thr-Asn, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val, Gly, Ala, or Asn optionally is replaced by —NH 2 .
8. The method of claim 6 , wherein Xaa2 is an analog of Trp comprising a substituted or unsubstituted bicyclic aromatic ring component or two or more substituted or unsubstituted monocyclic aromatic ring components.
9. The method of claim 6 , wherein Xaa2 is an analog of Trp comprising a lower alkoxy or lower alkyl substituent at the 1 or 5 position of tryptophan or a halogen substituent at the 5 or 6 position of tryptophan.
10. The method of claim 6 , wherein Xaa2 is an analog of Trp comprising a lower alkoxy or lower alkyl substituent at the 1 or 5 position of tryptophan or a halogen substituent at the 5 or 6 position of tryptophan and Xaa2* is Trp.
11. The method of claim 1 , wherein the compstatin analog comprises a cyclic peptide having a sequence selected from the group consisting of SEQ ID NOs: 14, 21, 28, 29, 32, 33, 34, or 36.