Methods and compositions related to peptides and proteins with C-terminal elements
Disclosed are compositions and methods useful for targeting and internalizing molecules into cells of interest and for penetration by molecules of tissues of interest. The compositions and methods are based on peptide sequences that are selectively internalized by a cell, penetrate tissue, or both. The disclosed internalization and tissue penetration is useful for delivering therapeutic and detectable agents to cells and tissues of interest.
1. A method of forming a CendR conjugate, the method comprising
(a) selecting an amino acid sequence comprising a CendR element for internalization into a cell, tissue penetration, or both, wherein the CendR element is at a C-terminal end of the conjugate and has an exposed C-terminal carboxyl group, wherein the CendR element has the sequence X 1 X 2 X 3 X 4 , where X 1 can be R, or K, where X 4 can be R, K, or KG, and where X 2 and X 3 can each be, independently, any amino acid, wherein X 1 and X 4 are not both R, and wherein the CendR element does not include KLRK (SEQ ID NO:168), KPPR (SEQ ID NO:169), KRSR (SEQ ID NO:170), KWKK (SEQ ID NO:173), RRLK (SEQ ID NO:178), RRTK (SEQ ID NO:183), RRPK (SEQ ID NO:184), KQRR (SEQ ID NO:185), KRAR (SEQ ID NO:186), KRGR (SEQ ID NO:187), RSFK (SEQ ID NO:188), KKPR (SEQ ID NO:189), RLAK (SEQ ID NO:174), or KRTR (SEQ ID NO:191), and
(b) causing a cargo composition to be covalently coupled or non-covalently associated with a protein or peptide comprising the selected amino acid sequence,
wherein the cargo composition is coupled or associated with the protein or peptide on the N terminal side of the CendR element,
wherein the CendR conjugate comprises the protein or peptide and the coupled or associated cargo composition,
wherein the CendR element binds to neuropilin 1 (NRP-1), wherein internalization of the CendR element into cells is dependent on NRP-1, and
wherein the CendR element has the sequence KLAK (amino acids 11-14 of SEQ ID NO:5), KPAR (amino acids 4-7 of SEQ ID NO:114), KPER (amino acids 4-7 of SEQ ID NO:90), KPLR (amino acids 4-7 of SEQ ID NO:63), KPRR (amino acids 5-8 of SEQ ID NO:126), KQAR (amino acids 4-7 of SEQ ID NO:64), KRDR (amino acids 3-6 of SEQ ID NO:89), KRER (amino acids 4-7 of SEQ ID NO:91), KTAR (amino acids 3-6 of SEQ ID NO:56), RGDK (amino acids 2-5 of SEQ ID NO:4), RPAK (amino acids 4-7 of SEQ ID NO:112), or RTPK (amino acids 4-7 of SEQ ID NO:84).
2. A CendR conjugate made by the method comprising
(a) selecting an amino acid sequence for internalization into a cell, tissue penetration, or both, wherein the amino acid sequence comprises a CendR element, wherein the CendR element is at a C-terminal end of the conjugate and has an exposed C-terminal carboxyl group, wherein the CendR element has the sequence X 1 X 2 X 3 X 4 , where X 1 can be R, or K, where X 4 can be R, K, or KG, and where X 2 and X 3 can each be, independently, any amino acid, wherein X 1 and X 4 are not both R, and wherein the CendR element does not include KLRK (SEQ ID NO:168), KPPR (SEQ ID NO:169), KRSR (SEQ ID NO:170), KWKK (SEQ ID NO:173), RRLK (SEQ ID NO:178), RRTK (SEQ ID NO:183), RRPK (SEQ ID NO:184), KQRR (SEQ ID NO:185), KRAR (SEQ ID NO:186), KRGR (SEQ ID NO:187), RSFK (SEQ ID NO:188), KKPR (SEQ ID NO:189), RLAK (SEQ ID NO:174), or KRTR (SEQ ID NO:191), and
(b) causing a cargo composition to be covalently coupled or non-covalently associated with a protein or peptide comprising the selected amino acid sequence,
wherein the cargo composition is coupled or associated with the protein or peptide on the N terminal side of the CendR element,
wherein the CendR conjugate comprises the protein or peptide and the coupled or associated cargo composition,
wherein the CendR element binds to neuropilin 1 (NRP-1), wherein internalization of the CendR element into cells is dependent on NRP-1, and
wherein the CendR element has the sequence KLAK (amino acids 11-14 of SEQ ID NO:5), KPAR (amino acids 4-7 of SEQ ID NO:114), KPER (amino acids 4-7 of SEQ ID NO:90), KPLR (amino acids 4-7 of SEQ ID NO:63), KPRR (amino acids 5-8 of SEQ ID NO:126), KQAR (amino acids 4-7 of SEQ ID NO:64), KRDR (amino acids 3-6 of SEQ ID NO:89), KRER (amino acids 4-7 of SEQ ID NO:91), KTAR (amino acids 3-6 of SEQ ID NO:56), RGDK (amino acids 2-5 of SEQ ID NO:4), RPAK (amino acids 4-7 of SEQ ID NO:112), or RTPK (amino acids 4-7 of SEQ ID NO:84).