A chemical approach to the pharmaceutical optimization of an anti-HIV protein.

Chemical protein synthesis is important for dissecting the molecular basis of protein function. Here we advance its scope by demonstrating the significant improvement of the multifaceted pharmaceutical profile of small proteins exclusively via a chemical-based approach. The focus of this work centered on CCL-5 (RANTES) derivatives with potent anti-HIV activity. The overall chemical strategy involved a combination of coded and noncoded amino acid mutagenesis, peptide backbone engineering, and site-specific polymer attachment. The ability to alter specific protein residues, as well as precise control of the position and type of polymer attachment, allows for the exploration of specific molecular designs and resulted in novel CCL-5 analogues with significant differences in their respective biochemical and pharmaceutical properties. Using this approach, the complex-interplay of variables contributing to the noncovalent self-association (aggregation) state, CCR-5 specificity, in vivo elimination half-life, and anti-HIV activity of CCL-5-based protein analogues could be empirically evaluated via total chemical synthesis. This work has led to the identification of potent (sub-nanomolar) anti-HIV proteins with significantly improved pharmaceutical profiles, and illustrates the increasing value of protein chemical synthesis in contemporary therapeutic discovery. These antiviral molecules provide a novel mechanism of action for the development of a new generation of anti-HIV therapeutics which are still desperately needed.

Medicinal chemistry applied to a synthetic protein: development of highly potent HIV entry inhibitors.

We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl1]RANTES(2-68), known as AOP-RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure-activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES [N-nonanoyl, des-Ser1[L-thioproline2, L-cyclohexylglycine3]-RANTES(2-68)], which is 50 times more potent than AOP-RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

A divergent INS protein in Caenorhabditis elegans structurally resembles human insulin and activates the human insulin receptor.

Caenorhabditis elegans contains a family of putative insulin-like genes proposed to regulate dauer arrest and senescence. These sequences often lack characteristic sequence features of human insulin essential for its folding, structure, and function. Here, we describe the structure and receptor-binding properties of INS-6, a single-chain polypeptide expressed in specific neurons. Despite multiple nonconservative changes in sequence, INS-6 recapitulates an insulin-like fold. Although lacking classical receptor-binding determinants, INS-6 binds to and activates the human insulin receptor. Its activity is greater than that of an analogous single-chain human insulin analog.

Design and chemical synthesis of a homogeneous polymer-modified erythropoiesis protein.

We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.

Sex-specific gene regulation. The Doublesex DM motif is a bipartite DNA-binding domain.

Sex-specific gene expression in Drosophila melanogaster is regulated in part by the Doublesex (DSX) transcription factor. Male- and female-specific splicing isoforms share a novel DNA-binding domain, designated the DM motif. This domain is conserved among a newly recognized family of vertebrate transcription factors involved in developmental patterning and sex determination. The DM motif consists of an N-terminal zinc module and a disordered C-terminal tail, hypothesized to fold on specific DNA binding as a recognition alpha-helix. Truncation of the tail does not perturb the structure of the zinc module but impairs DNA binding and DNA-dependent dimerization. Chemical protein synthesis and alanine scanning mutagenesis are employed to test the contributions of 13 side chains to specific DNA binding. Selected arginine or lysine residues in the zinc module were substituted by norleucine, an isostere that maintains the aliphatic portion of the side chain but lacks a positive charge. Arginine or glutamine residues in the tail were substituted by alanine. Evidence is obtained that both the zinc module and C-terminal tail contribute to a bipartite DNA-binding surface. Conserved arginine and glutamine residues in the tail are required for high affinity DNA recognition, consistent with its proposed role as a nascent recognition alpha-helix.