Chemical Peptide Synthesis in the Development of Protein Therapeutics

Transcription

1 Chemical Peptide Synthesis in the Development of Protein Therapeutics Stephen Kent Abstract Effective automated SPPS is key to the synthesis of peptide building blocks for chemical protein synthesis, and for the production of peptides and small proteins for pre-clinical studies. In the research laboratory, the total synthesis of proteins based on modern chemical ligation methods has enabled the use of mirror image protein molecules for the determination of X-ray structures by racemic crystallography. Facile chemical synthesis of D-protein therapeutic targets, in combination with mirror image phage display, promises the development of a novel class of small D-protein therapeutics that will be produced by chemical manufacture. 1. Chemical protein synthesis The total synthesis of proteins based on modern chemical ligation methods has become a versatile and effective research tool. Numerous proteins, ranging in size up to more than 200 amino acid residues, have been synthesized in native, fully functional form. In addition, total synthesis has enabled the preparation of unique chemical protein analogues and the site-specific incorporation of spectroscopic probe moieties into the protein molecule. Modern chemical protein synthesis is based on a set of underlying technologies, as shown in Scheme 1. Unprotected Peptide Segments Chemoselective & Regiospecific Covalent Condensation of Unprotected Peptide Segments Large Polypeptide Chemical Ligation Underlying Technologies: Solid Phase Peptide Synthesis Electrospray MS Protein NMR X-ray Crystallography Scheme 1. Modern chemical protein synthesis [Ref. 1] Protein Molecule In addition to the chemical ligation of unprotected peptide segments, a key enabling technology is the facile preparation of unprotected peptide segment building blocks by highly optimized solid phase peptide synthesis. These building blocks consist of Cys-peptides and peptide-thioesters. The Cys-peptides may be made by the widely used Fmoc chemistry SPPS or by Boc chemistry SPPS. However, the robust preparation of peptide-thioesters is still limited to the use of in situ neutralization Boc chemistry SPPS; recent advances in the synthesis of peptidethioesters by Fmoc SPPS make use of an ingenious novel linker chemistry (Figure 1) and promise the routine preparation of peptide-thioesters by this more widely used Fmoc chemistry Recent developments in chemical protein synthesis Kinetically controlled ligation of unprotected peptides has enabled the total synthesis of larger protein molecules in a straightforward fashion. In conjunction with native chemical ligation, kinetically controlled ligation has been used for the fully convergent chemical synthesis of polypeptide chains of more than 200 amino acid residues. For example, it was possible to make a covalent dimer form of the HIV-1 protease molecule comprising a polypeptide of 203 amino acids, with full enzyme activity; high resolution X-ray diffraction confirmed the crystal structure of the synthetic enzyme molecule (Figure 2). PharManufacturing: The International Peptide Review

2 Figure 1. Chemistry for the Fmoc SPPS of peptide-thioesters [Ref. 2]. Figure 2. Covalent dimer HIV-1 protease prepared by total chemical synthesis [Ref. 1]. PharManufacturing: The International Peptide Review 17

3 3. Racemic protein crystallography for the determination of X-ray structures of novel proteins Chemical synthesis by modern ligation methods has enabled the facile preparation of mirror image protein molecules. A D-protein molecule has a three dimensional (tertiary) structure that is in every respect the mirror image of the structure of the natural L-protein structure. The chemical synthesis of D-proteins was pioneered in the early 1990s, but remained a laboratory curiosity until recently when it was shown that the use of a racemic protein mixture could facilitate the crystallization of recalcitrant proteins for the determination of novel molecular structures by X-ray crystallography (Figure 3). In the past two years, racemic protein crystallography enabled by chemical protein synthesis has been used to solve the structures of a series of proteins of ever increasing size. The scope and limitations of the racemic protein crystallography method are still being investigated. 4. Mirror image proteins in drug discovery Access to mirror image proteins enabled by total chemical synthesis has stimulated commercial interest in using these unique versions of therapeutic targets to develop novel classes of drug molecules. The fundamental concept is to screen libraries of chiral binders against the mirror image of the target therapeutic protein molecule, and to then use chemistry to make the mirror image of the selected binders which will be active against the natural form of the therapeutic target protein. The process is illustrated for peptides in Scheme 2. Figure 3. Racemic crystallography of snow flea antifreeze protein [Ref. 3]. Scheme 2. Mirror image phage display [Ref. 4]. Several companies are active in this area of drug discovery. Berlin-based Noxxon Pharma AG is developing mirror image nucleic acid molecules, known as spiegelmers, as drugs for the treatment of inflammatory diseases and hematological indications. The Braunschweig-based company Cosmix is focused on using RNA display technology to develop D-peptide drugs. Reflexion Pharmaceuticals is a San Francisco-based company that is focused on using mirror image phage display to develop D-protein drugs. The use of mirror image proteins in drug discovery has been validated by the fact that Noxxon now has products in Phase 1 clinical trials - Cosmix and Reflexion are in pre-clinical development Mirror image phage display In 1997 Peter Kim of the Whitehead Institute at MIT developed mirror image phage display, a method for systematically developing D-peptide ligands for natural L-proteins as candidate therapeutics (Scheme 2). Mirror image phage display depends on the use of the D-protein form of a target molecule for panning a peptide phage library; the identified L-peptide ligands for the D-protein are then chemically synthesized in D-form. The resulting D-peptides are obligate ligands for the natural L-protein, with identical specificity and affinity. D-Peptides have properties that are highly desirable in candidate therapeutics: they are non-immunogenic, are completely resistant to the action of natural endo- and exo-proteinases, and are silent to metabolism and consequently are secreted intact in the urine. PharManufacturing: The International Peptide Review

4 Given the potential advantages of D-peptide and D-protein therapeutics, why has mirror image page display not been used more widely? The bottleneck in the routine application of mirror image phage display is access to the D-form of the target protein: until the advent of modern chemical ligation methods, and in particular the invention of native chemical ligation for the total chemical synthesis of native protein molecules, it simply was not possible to make the typically 200+ residue target proteins by total chemical synthesis. In the past few years, that situation has changed. Armed with the combination of kinetically controlled ligation and native chemical ligation, it has proved possible to prepare the functional form of VEGF-A in both L- and D-protein forms, and to determine the X-Ray structures of both forms. VEGF-A is the target of many of the therapeutic antibodies that form the fastest growing class of human drugs, and as such is a compelling target for mirror image phage display (Scheme 3). Scheme 3. VEGF-targeted mirror image phage display [Ref. 5]. Note that it is still imperative to be able to make high purity peptide-thioester building blocks in order to make D-protein targets. Because the Boc chemistry SPPS necessary for making peptide-thioesters has fallen into disuse worldwide, this represents another barrier to the widespread use of mirror image phage display. 6. Automated peptide synthesizer instruments The facile total synthesis of protein molecules that is made possible by modern chemical ligation methods depends on the ability to make high purity Cys-peptides and peptidethioesters in good yield. Machine-assisted ( automated ) peptide synthesis has an essential contribution to make to the synthesis of unprotected peptide building blocks. Either Fmoc or Boc SPPS can be used to make peptides with an N-terminal Cys residue. However, peptide-thioesters are best made by Boc in situ neutralization chemistry SPPS. Thus, there is a need for a versatile automated peptide synthesizer instrument that can make peptides by either Boc or Fmoc chemistry SPPS. Another important feature of a practical automated peptide synthesizer is that it must be possible for the researcher to repair it easily and at nominal cost. Most commercially available peptide synthesizers are over-engineered with the essentially Figure 4. The CS Bio model CS336X automated laboratory peptide synthesizer. futile aim of preventing failures: the corrosive chemicals used in SPPS inevitably lead to valve and other failures, regardless of the sophistication of the engineering in the instrument being used. What is needed is a simple peptide synthesizer that can be used for either Fmoc or Boc chemistry SPPS and that is simple and inexpensive to repair. The CS Bio line of automated synthesizers fits this bill of particulars best of currently commercial peptide synthesizer instruments (Figure 4). PharManufacturing: The International Peptide Review 19

5 In addition to providing peptide building blocks for chemical synthesis of larger proteins, automated peptide synthesizers can greatly facilitate the solid phase synthesis of peptides and small proteins for pre-clinical studies. Typically, only limited amounts of the candidate therapeutic peptide or small protein are required at this stage of product testing. Machine-assisted SPPS has the great advantage of enabling the straightforward, rapid development of well-documented methods for the synthesis of multi-gram quantities of peptides for pre-clinical studies. 7. Chemical manufacture of protein therapeutics Protein therapeutics, particularly engineered antibodies, are the most rapidly growing class of human therapeutics. This despite the fact that the development and particularly the manufacture of therapeutic antibodies is hugely expensive. Current estimates for the cost of construction of a recombinant antibody manufacturing plant are in the range of $200+ million dollars. Furthermore, the use of chemistry to modify the pharmacokinetics and other properties of proteins therapeutics is difficult or impossible to effect with recombinant proteins. It would be highly desirable to use chemical synthesis to manufacture protein therapeutics. What are the prospects for the chemical manufacture of protein therapeutics? An important recent development in the chemical synthesis of peptides is the widespread adoption by the industry of hybrid SPPS-solution methods for the scale manufacture of large polypeptides. The hybrid method (Scheme 4) was pioneered by Kleomenis Barlos, and was employed for the manufacture of the 36 amino acid residue peptide therapeutic Fuzeon at large scale. Chemical manufacture at the metric ton scale entailed optimization of the supply chain of chemicals used in the synthesis of protected peptides by Fmoc chemistry SPPS, as well as the use of robust and inexpensive purification methods. These developments in large scale manufacture of long peptides have been described. Hybrid SPPS/solution synthesis can be used for the manufacture of polypeptides up to ~60 amino acids in length. Some existing protein therapeutics, as well as small proteins under development as candidate therapeutics, comprise ~40-60 amino acid residues, and may contain multiple disulfide bonds. These small protein therapeutics can be manufactured at scale by established chemical synthesis methods. Chemical manufacture can be carried out under CGMP by existing contract manufacturers, without the costs involved in setting up dedicated manufacturing plants from scratch for each therapeutic product. In this respect, chemical manufacture offers a significant advantage over recombination manufacture for small protein therapeutics. Scheme 4. The hybrid method for the manufacture of long peptides [Ref.6]. 20 Human insulins are a particularly important class of human therapeutics. The development of insulins with improved properties in terms of speed and duration of action and reduced tendency to aggregation has significantly improved diabetes drug therapy over the past ~15 years. These insulin analogue therapeutics are coming off patent starting in the next few years, and chemical manufacture of generic forms of these drugs could result in wider availability of improved therapeutics at reduced cost to the patient. Until now, cost-effective chemical manufacture of human insulins has been prevented by the very low yields of chain combination, with concomitant formation of native disulfide bonds, starting from isolated insulin A and B chains. A solution to this long standing problem has recently been reported (Figure 5), and promises to render practical the cost-effective scale chemical manufacture of human insulins for therapeutic use. PharManufacturing: The International Peptide Review

6 Figure 5. Chemical synthesis of ester insulin and its conversion to human insulin [Ref. 7]. 8. Chemical analogs of protein therapeutics A major advantage of chemical manufacture for the production of peptide and protein therapeutics is the inherent ability to make precise modifications, at will, in order to tune the properties of the protein molecule. Chemical modifications, such as the attachment of PEG or other polymers, can be used to modify the immunogenicity of an L-peptide or small L-protein therapeutic molecule. In addition, acetylation of the N-terminal and incorporation of a C-terminal carboxamide moiety can greatly decrease the susceptibility to proteolytic digestion, and thus affect both the immunogenicity and PK properties of the molecule. For small D-proteins, the chemical attachment of PEG-like polymers can be used to systematically tune the PK properties of a candidate therapeutic molecule. Author Profile Stephen Kent is on the faculty of the University of Chicago, and is a co-founder of Reflexion Pharmaceuticals References 1. Total chemical synthesis of proteins, Kent, S.B., Chemical Society Reviews, 38, (2009). 2. An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation, Blanco-Canosa J.B., Dawson P.E., Angew. Chem. Int. Ed., 47, (2008). 3. X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers, Pentelute B.L., et al., J Am Chem Soc, 130, (2008). 4. Mirror image phage display a method to generate D-peptide ligands for use in diagnostic or therapeutical applications, Funkea S.A., Willbold D., Mol. BioSyst., 5, (2009). 5. Courtsey of Reflexion Pharmaceuticals 6. Development of HIV fusion inhibitors, Schneider S.E., et al., J. Peptide Sci., 11, (2005) 7. Design and total synthesis of [GluA4(OβThrB30)]insulin ( ester insulin ): a minimal proinsulin surrogate that can be chemically converted into human insulin, Sohma Y., et al., Angewandte Chemie, 49, in press (2010). PharManufacturing: The International Peptide Review 21