1. Discovery of a novel nonphosphorylated pentapeptide motif displaying high affinity for Grb2-SH2 domain by the utilization of 3'-substituted tyrosine derivatives
Yan-Li Song, Megan L Peach, Peter P Roller, Su Qiu, Shaomeng Wang, Ya-Qiu Long J Med Chem. 2006 Mar 9;49(5):1585-96. doi: 10.1021/jm050910x.
The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that represents an attractive target for anticancer therapeutic intervention. An impressive number of synthetic Grb2-SH2 domain inhibitors have been identified; however, clinical agents operating by this mechanism are lacking, due in part to the unique requirement of anionic phosphate-mimicking functionality for high SH2 domain-binding affinity or the extended peptide nature of most inhibitors. In the current study, a new binding motif was successfully developed by the incorporation of 3'-substituted tyrosine derivatives into a simplified nonphosphorylated cyclic pentapeptide scaffold (4), which resulted in high affinity Grb2-SH2 inhibitors without any phosphotyrosine or phosphotyrosine mimetics. The new L-amino acid analogues bearing an additional nitro, amino, hydroxy, methoxy or carboxy group at the 3'-position of the phenol ring of tyrosine were prepared in an orthogonally protected form suitable for solid-phase peptide synthesis using Fmoc protocols. The incorporation of these residues into cyclic peptides composed of a five-amino acid sequence motif, Xx(1)-Leu-(3'-substituted-Tyr)-Ac6c-Asn, provided a brand new class of nonphosphorylated Grb2 SH2 domain inhibitors with reduced size, charge and peptidic character. The highest binding affinity was exhibited by the 3'-aminotyrosine (3'-NH2-Tyr)-containing (R)-sulfoxide-cyclized pentapeptide (10b) with an IC50 = 58 nM, the first example with low-nanomolar affinity for a five-amino acid long sequence binding to Grb2-SH2 domain free of any phosphotyrosine or phosphotyrosine mimics. However, the incorporation of 3'-NO2-Tyr, 3'-OH-Tyr or 3'-OCH3-Tyr surrogates in the pentapeptide scaffold is detrimental to Grb2-SH2 binding. These observations were rationalized using molecular modeling. More significantly, the best Grb2-SH2 inhibitor 10b showed excellent activity in inhibiting the growth of erbB2-dependent MDA-MB-453 tumor cell lines with an IC50 value of 19 nM. This study is the first attempt to identify novel nonphosphorylated high affinity Grb2 SH2 inhibitors by the utilization of 3'-substituted tyrosine derivatives, providing a promising new strategy and template for the development of non-pTyr-containing Grb2-SH2 domain antagonists with potent cellular activity, which potentially may find value in chemical therapeutics for erbB2-related cancers.
2. Solid-Phase Total Synthesis of Bacitracin A
Jinho Lee, John H. Griffin, Thalia I. Nicas J Org Chem. 1996 Jun 14;61(12):3983-3986. doi: 10.1021/jo960580b.
An efficient solid-phase method for the total synthesis of bacitracin A is reported. This work was undertaken in order to provide a general means of probing the intriguing mode of action of the bacitracins and exploring their potential for use against emerging drug-resistant pathogens. The synthetic approach to bacitracin A involves three key features: (1) linkage to the solid support through the side chain of the L-asparaginyl residue at position 12 (L-Asn(12)), (2) cyclization through amide bond formation between the alpha-carboxyl of L-Asn(12) and the side chain amino group of L-Lys(8), and (3) postcyclization addition of the N-terminal thiazoline dipeptide as a single unit. To initiate the synthesis, Fmoc L-Asp(OH)-OAllyl was attached to a PAL resin. The chain of bacitracin A was elaborated in the C-to-N direction by sequential piperidine deprotection/HBTU-mediated coupling cycles with Fmoc D-Asp(OtBu)-OH, Fmoc L-His(Trt)-OH, Fmoc D-Phe-OH, Fmoc L-Ile-OH, Fmoc D-Orn(Boc)-OH, Fmoc L-Lys(Aloc)-OH, Fmoc L-Ile-OH, Fmoc D-Glu(OtBu)-OH, and Fmoc L-Leu-OH. The allyl ester and allyl carbamate protecting groups of L-Asn(12) and L-Lys(8), respectively, were simultaneously and selectively removed by treating the peptide-resin with palladium tetrakis(triphenylphosphine), acetic acid, and triethylamine. Cyclization was effected by PyBOP/HOBT under the pseudo high-dilution conditions afforded by attachment to the solid support. After removal of the N-terminal Fmoc group, the cyclized peptide was coupled with 2-[1'(S)-(tert-butyloxycarbonylamino)-2'(R)-methylbutyl]-4(R)-carboxy-Delta(2)-thiazoline (1). The synthetic peptide was deprotected and cleaved from the solid support under acidic conditions and then purified by reverse-phase HPLC. The synthetic material exhibited an ion in the FAB-MS at m/z 1422.7, consistent with the molecular weight calculated for the parent ion of bacitracin A (MH(+) = C(73)H(84)N(10)O(23)Cl(2), 1422.7 g/mol). It was also indistinguishable from authentic bacitracin A by high-field (1)H NMR and displayed antibacterial activity equal to that of the natural product, thus confirming its identity as bacitracin A. The overall yield for the solid-phase synthesis was 24%.
3. L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides
B Ye, M Akamatsu, S E Shoelson, G Wolf, S Giorgetti-Peraldi, X Yan, P P Roller, T R Burke Jr J Med Chem. 1995 Oct 13;38(21):4270-5. doi: 10.1021/jm00021a016.
Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.
