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Direct Sequence Conversion 8.2.1.1 RNA-binding Peptoids

Transcription of HIV RNA requires the interaction of the virally encoded Tat protein with the transcriptional activator-responsive (TAR) element [157], a bulged RNA hairpin structure. A single Arg amidst at least three basic residues in the Arg-rich region of the Tat protein is the key determinant for binding the trinucleotide bulge in TAR RNA [158], An a-peptide based on tat with the sequence Ac-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH2 was found to bind TAR RNA with submicromolar affinity (KD = 780 nM) based on electrophoretic mobility shift assays [158]. Furthermore, circular dichroism studies with TAR RNA showed a characteristic signal decrease at 265 nm upon addition of Tat peptide, indicating a conformational change of TAR RNA upon peptide binding [159].

Various peptoids have been designed to specifically target TAR RNA (Fig. 8.6) [159, 160]. Peptoids based on the sequence of the Arg rich region of Tat had similar affinities compared with the Tat protein (@2 mM for peptoid) by gel shift mobility assay [159]. The CD spectrum for wild type TAR RNA exhibited the characteristic change for binding upon adding the peptoid, while no such change was observed for mutant TAR RNA, demonstrating high specificity for targeting TAR RNA. Also, fluorescence resonance energy transfer (FRET) experiments showed a high affinity complex between a peptoid amide analog and TAR RNA with sub-micromolar affinity (KD = 155 nM). An ester derivative of the same peptoid demonstrated even higher affinity (KD = 68 nM) (see Fig. 8.6) [160].

Fig. 8.6 TAR RNA binding (A) peptoids, and (B) oligocarbamate and oligourea designs.

Oligourea Oligocarbamate

Fig. 8.6 TAR RNA binding (A) peptoids, and (B) oligocarbamate and oligourea designs.

8.2.1.2 RNA-binding Oligourea and Carbamate

An oligourea was designed to bind HIV-1 TAR RNA by direct sequence conversion of the Arg-rich region of the Tat protein (Fig. 8.6) [161]. The oligourea bound the TAR RNA with submicromolar affinity (KD = 111 nM), similar to a Tat-derived peptide (KD = 780 nM) as determined by electrophoretic mobility shift assay [161]. Furthermore, mutant TAR RNA could not displace the oligourea from labeled TAR RNA, demonstrating the specificity of the oligourea for binding TAR RNA [161]. Also the oligourea did not bind TAR RNA with mutations immediately surrounding the pyrimidine bulge critical for recognition. Importantly, the oligourea was determined to be protease resistant against proteinase K. Similar results have been obtained for a carbamate derivative (Fig. 8.6) [162]. The oligocarbamate was found to bind TAR RNA with micromolar affinity (KD = 1.13 mM) as determined by mobility shift assay. Competition experiments on photo

Fig. 8.7 TAR RNA binding b-peptide designs.

crosslinked carbamate-RNA complexes revealed that carbamate binding was specific to the widened major groove of TAR RNA and required the presence of the characteristic trinucleotide bulge [162]. Treatment of the carbamate-RNA complex with proteinase K did not result in a loss of the photo crosslink, indicating much higher proteolytic stability compared with the Tat protein-RNA complex.

8.2.1.3 RNA-binding b-Peptides

TAR RNA-binding b3-peptides have also been designed as potential HIV therapeutics by direct sequence conversion of a segment of the RNA-binding protein Tat to the b-peptide sequence (Fig. 8.7) [163]. This 11-residue b3-peptide based on the Arg-rich region of the Tat protein bound TAR RNA with nanomolar affinity (KD = 29 nM) as determined by fluorescence anisotropy [163].

8.2.1.4 Receptor-binding b-Peptides

Somatostatin receptors bind somatostatin, a natural disulfide linked 14-residue cyclic a-peptide, to control the release of various hormones including growth hormone, glucagon, insulin, and gastrin [164]. Various somatostatin receptor subtypes for mediating the different biological activities have been identified [164]. Therefore, developing molecules that selectively target these receptors would be desirable for therapeutic purposes [164]. Somatostatin and most a-peptide somatostatin analogs adopt a b-turn conformation involving the amino acid sequence Phe-Trp-Lys-Thr (Fig. 8.8) [165]. The central Trp-Lys is required for binding soma-tostatic receptors and thus bioactivity [166, 167]. Octreotide (Sandostatin) is an 8-residue cyclic a-peptide somatostatin analog that may be used for the treatment of acromegaly and certain gastric-entero tumors (Fig. 8.8) [167, 168]. However, octreotide has a relatively short half life of 90 min in vivo, making b-peptides with increased bio-availability an attractive alternative.

Design efforts toward somatostatin receptor binding b-peptides have been based on the placement of somatostatin residues onto a b-peptide scaffold that can present the four bioactive side chains in a productive manner (Fig. 8.8). Initially, cyclic b-peptide tetramers were employed to present the side chains of the key residues of somatostatin Phe-Trp-Lys-Thr [149, 150]. Computational modeling showed that the constraints imposed by the cyclic structure matched reasonably well with the type II0 b-turn of a-peptides. The cyclic b-peptides bound different types of human somatostatin receptors with micromolar affinities (KD = 3.3-

Cyclic (3-peptide inhibitor Linear (3-peptide inhibitor

Fig. 8.8 The b-turn motif of SRIF-14, the somatostatin receptor inhibitor octreotide, a cyclic b-peptide somatostatin analog, and a linear b-peptide somatostatin analog.

Cyclic (3-peptide inhibitor Linear (3-peptide inhibitor

Fig. 8.8 The b-turn motif of SRIF-14, the somatostatin receptor inhibitor octreotide, a cyclic b-peptide somatostatin analog, and a linear b-peptide somatostatin analog.

186 mM). Interestingly, unconstrained linear b-peptides, that adopt a turn conformation, bound somatostatin receptors with nanomolar affinities (KD = 83-724 nM) [151, 169], suggesting that the constraints imposed by the cyclic b-peptide were less than optimal.

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