By Waleed Danho, PhD, Director, Boulder Peptide Society
Protein therapeutics is gaining headway in the treatment and prevention of a variety of maladies. Critical to the success and growing use of biologic-level pharmaceuticals is the development of methods for their precise synthesis. There are emerging technologies for the total synthesis of proteins, with a special focus on the possibility of accessing therapeutic targets through “chemoselective peptide ligations” (NCL)
In the recent December 2013 issue of SCIENCE (Vol.342, Page 1357-1360), Samuel J. Danishefsky et.al published an article entitled “Erythropoieten Derived by Chemical Synthesis”. Dr. Danishefsky states “The total synthesis of a homogeneous erythropoietin (EPO), possessing the native amino acid sequence and chitobiose glycans at each of the three wild-type sites of N glycosylation, has been accomplished. We provide herein an account of our decade-long research effort en route to this formidable target compound. The optimization of the synergy of the two bedrock sciences we now call biology and chemistry was central to the success of the synthesis of EPO”.
The Chemoselective peptide ligation based on the pioneering work of Stave Kent et al., was published in Science 1994 (P.E. Dawson, T.W. Muir, I. Clark,-Lewis, and S.B. Kent Science 266, 776-779 ( 1994). This method enables the efficient total chemical synthesis of enzymes and other protein molecules. The ideal is the chemo selective reaction of unprotected synthetic peptides. The native chemical ligation enables the reaction of two or more unprotected peptides in aqueous solution at neutral pH to form a single product in near quantitative yield. Full-length synthetic polypeptides are folded to form the defined tertiary structure of the target protein.
Erythropoietin (EPO) is a 166 –amino acid glycoprotein hormone that regulates the production of red blood cells. A major challenge to obtaining pure, homogenous EPO is that the cells produce the protein as a mixture of glycosylated forms (glycoforms). The protein undergoes extensive and variable glycosylation, at four distinct sites (Serine 126, Asparagin 24, 38, and 83). The commercial analogs of EPO, Pocrit (Janssen), Epogen (Amgen), and Aransep (Amgen) are also as a mixture of glycoforms, Studies suggests that the specific sugar modification can affect the stability and activity of EPO. Thus systematic investigation into the structure-activity relations of the sugars and targeted production of the most active glycoform could improve the efficacy of EPO and other biologics.
Danishefsky’s group tackles this problem by using (NCL) and succeeds in the total synthesis of homogenous EPO glycoform 1. The fully synthetic EPO glycoform exhibits enhanced stability relative to it’s counterpart-smaller-glycans, and upon folding, displays activity in vivo comparable to that of the clinical drug Procrit.
Further advances are still required to be able to compete with the biological methods of EPO production in terms of speed, and scalability and production. Nevertheless, the chemical synthesis of such a complex molecule represents an impressive accomplishment by Danishefsky’s lab. and validation for the NCL methodology by Kent et al. and could boost many other protein -based drugs as well.