Peptide based drug discovery and research is increasingly at the forefront in addressing new therapeutic challenges due to their high target selectivity and potency with low toxicity. Automated solid phase peptide synthesis (SPPS) increases reliability, efficiency, and crude purity for peptides in drug discovery and development. For example, introducing heat (>50°C) in the SPPS of linear peptides has shown improved results with shorter coupling cycles and higher crude purity.
The MK2 inhibitor, MMI-0100, is a cell penetrating peptide that is currently undergoing clinical trials for fibrotic and inflammatory lung disease [1,2]. Previously, the optimized synthesis of a MMI-0100 analog (AAVGLQRALAKARAQRAAARAY) was done using an automated system with a four-day method that consisted of double and triple couplings with the highest purities around 50%. In this study, different conditions and methods for the synthesis of the MMI-0100 analog using parallel synthesis were assessed. The full synthesis was completed in less than 24 h with improved crude purities.
 Komalavilas, Am J Respir Crit Care Med, vol. 193, p. A1349, 2016.
 "Moerae Matrix - Scientific Platform," Moerae Matrix, 2017. [Online]. Available: http://moeraematrix.com/. [Accessed March 2017].
Purification of crude synthetic peptide mixtures often employs a multi-step chromatographic purification process. The first step removes most of the undesired components, followed by a different step to “polish” the material to the desired purity level. If applicable, a single step process can produce significant time and cost savings provided the single step can achieve the necessary purity while maintaining a desirable yield and throughput. A multistep process using the same stationary phase, can provide savings of time and costs.
The work presented here demonstrates the development of a multi-step purification process on a single stationary phase for a commercially significant crude synthetic peptide mixture, Exenatide (Figure 1.). The focus is on the initial development work, including the screening of multiple conditions to evaluate which steps will produce material of suitable purity. The investigated parameters include eluent pH, buffer components, and organic solvent composition.
A sensitive and specific LC-MS/MS assay for the determination of SPR741 has been developed and validated in four matrices. A Waters™ Oasis weak cation exchange SPE plate isolates SPR741 and its isotopic internal standard with high recovery and significant removal of matrix interferences. Binding to plastic is mitigated through pH control and non-specific binding losses from urine are controlled by the use of acidified CHAPS. The methods are precise and accurate across a 1000-fold range, and have been successfully employed for regulated analysis of hundreds of preclinical samples.
The ARGEN, by Fluence Analytics, provides researchers the tools to quickly detect changes in the normalized molecular weight of peptide samples in solution, enabling them to identify the onset of aggregation, degradation or phase changes while these phenomena take place. The ARGEN does this by continuously monitoring multiple independent static light scattering experiments, each one with its own set of controlled stressor conditions. These ARGEN controlled stressor conditions include both temperature from 18 oC – 100 oC as well as stirring from 0 – 2000 RPM but researchers can additionally screen many common formulation conditions for each sample by adjusting the solvent, pH, salt type and concentration and excipient type and concentration.
The data presented, shows how ARGEN was used to study the stability of peptides as influenced by pH, sample concentration, temperature and stirring stress in real time. This information can be used to preemptively understand how and when aggregation or degradation may occur so that additional formulation development can be focused to minimize these unwanted results.
We recently identified a new class of cyclic peptide triazoles that can irreversibly inactivate the HIV-1 envelope (Env) trimer on virus and Env-expressing cells. Linear peptide triazoles (PTs) were previously identified that inactivate HIV-1 Env by binding to the conserved CD4 binding epitope on Env-gp120. PT binding leads to inhibition of further conformational changes in the Env to achieve co-receptor binding and therefore halts the entry process. The irreversible inactivation process occurs as a result of efficient gp120 shedding of the virions upon binding to PTs, leaving only gp41-coated virions that are no longer infectious. Through side chain-to-side chain cyclization and chemical modifications including on-resin C-C coupling, we generated a class of cyclic peptide triazoles (cPTs) that, in addition to retaining all phenotypes of the linear peptides, massively resist proteolytic degradation and show promising in vivo half-life of ~ 3 h in rats. Both linear and cyclic peptide triazoles also bind to envelope on infected cells and cause gp120 shedding similar to that observed with the virions, suppressing the formation of new infectious viruses from infected cells. Selective killing of HIV-1 infected cells has been successfully demonstrated by multivalent linear PTs. Recapitulating this Env-targeting inactivation with the more advanced cPTs, and combining Env with strategies for infected cell activation and Env expression, offer the potential to use the cPT class of inhibitors in HIV CURE development.
Bicyclic RGD-peptides with high affinity and selectivity for αvβ3, αvβ5, and α5β1 integrins
D. Bernhagen[a], L. De Laporte[b], P. Timmerman[a,c]
[a] Pepscan Therapeutics, Zuidersluisweg 2, 8243 RC Lelystad, Netherlands; [b] DWI – Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany; [c] Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, Sciencepark 904 XH Amsterdam, Netherlands
The ‘bicyclic peptide’ platform recently attracted considerable interest as a powerful platform for novel therapeutic drugs because of their high binding affinities and selectivities in combination with an appreciable proteolytic stability. So far, only few cellular proteins have been evaluated for their binding to bicyclic peptides. The integrins represent an interesting target class for novel therapeutic agents because of their significant role in key pathological processes. We therefore synthesized libraries of hundreds of different bicyclic peptides, one loop comprising the well-known “RGD”-motif and providing basic integrin-affinity, and second loop consisting of a random sequence (XXX) that allows for controlling the binding selectivity. In order to screen for affinity and selectivity to each integrin (αvβ3, αvβ5, and α5β1) we developed a robust and cost-effective screening assay using a knottin-RGD peptide as a reference binder.[4,5] After selection of the best binders for each integrin, we designed 2nd and 3rd generation diversity libraries and thus gradually improved the affinity and selectivity for each integrin. The best IC50 values that we obtained in this way were, for example, 30 nM for αvβ3 (GRGDS: 5 µM, knottin-RGD: 250 nM), and 90 nM for α5β1 (GRGDS: >30 µM, knottin-RGD: 100 nM). We also labeled the best bicyclic integrin-binders with a fluorescent dye and studied their integrin-binding on live cells. The most potent bicyclic integrin-binders showing highest were also immobilized on 2D hydrogels in order to investigate their effect on cell adhesion, cell proliferation and viability as compared to the conventional RGD-peptides.
 “Astrazeneca takes Bicycle to work in potential $1B multitarget collaboration”, Bioworld™ Today 2016, 27, 3.
 P. Li, P. P. Roller, Curr. Top. Med. Chem. 2002, 2, 325–341; V. Baeriswyl, C. Heinis, ChemMedChem 2013, 8, 377–384.
 M. Barczyk, S. Carracedo, D. Gullberg, Cell Tissue Res. 2010, 339, 269–280; Y. Takada, X. Ye, S. Simon, Genome Biol. 2007, 8, 215.
 D. Bernhagen, L. De Laporte, P. Timmerman, Anal. Chem. 2017, 89, 5991–5997.
 R. H. Kimura, A. M. Levin, F. V Cochran, J. R. Cochran, Proteins 2009, 77, 359–369.
Cole W. Seifert and Guigen Li
GAP Peptides LLC, Lubbock, TX 79415, United States; Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, United States; Institute of Chemistry and Biomedical Sciences, Nanjing University, Nanjing 210093, P. R. China.
The peptide therapeutics market has been growing rapidly for years, both financially and as an increasing sector of the pharmaceutical industry.1 This market is projected to continue growing; therefore, new synthetic methodologies to reduce the cost of peptide synthesis and enable larger scale syntheses are highly desired.2 We have developed a solution-phase, Fmoc / tBu peptide synthesis method utilizing Group-Assisted Purification (GAP) chemistry, by design of a small-molecule (MW = 292 Da) protecting group. This protecting group, equipped with a phosphine oxide moiety, facilitates selective precipitation of the peptide from solution after each coupling step, with an average isolated yield per coupling of 97%. All fully protected peptides are white, crystalline solids that are easy to handle and can be stored for over 1 year without noticeable degradation. Reactions are run in homogeneous solution without the use of bulky polymers, leading to highly efficient couplings and high crude peptide purity. GAP peptide synthesis is amenable to a wide variety of coupling strategies, protecting groups, and deprotection reagents. Our process is also semi-automated, and we are working towards full automation. Examples of peptides synthesized using the GAP process are presented.
1. Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D., The Future of Peptide-based Drugs. Chem. Biol. Drug Des. 2013, 81 (1), 136-147.
2. Fosgerau, K.; Hoffmann, T., Peptide therapeutics: current status and future directions. Drug Discov. Today 2015, 20 (1), 122-128.
Structure-rich Phylomer peptide libraries, derived from biodiverse genomes, are a source of new cell penetrating peptides (CPPs) able to deliver therapeutic macromolecule cargoes to the intracellular space, thus opening up the druggable landscape. Employing a novel genetic screen known as the ‘endosome escape trap’ rare Phylomer CPPs were isolated that were able to deliver 60nm phage nanoparticle cargoes to the cytoplasm. These new cell penetrating Phylomers known as ‘FPPs’ show no evidence of toxicity and can be targeted to particular cell types using receptor binding domains. A variety of functional assays have been used to determine the extent of delivery of peptide and protein cargoes to the cytoplasm or nucleus. One such assay, based on split GFP complementation, has shown Phylomer FPPs to be 37-160 times more efficient at cytoplasmic delivery than conventional CPPs such as TAT or R9. Phylomer FPPs have also been shown to effectively deliver a 90 amino acid MYC inhibitor in a mouse model of breast cancer. This new delivery approach is being applied in conjunction with Phylomer peptides targeting transcription factor oncoproteins such as cMyc, MycN and STAT5, for more efficient cell targeted intracellular delivery of protein conjugates of more than 50 kDa in size, and also for intracellular delivery of nucleic acids.
Small molecules are only able to target the small population of human proteins containing a hydrophobic pocket which is a major problem in traditional small molecule drug development. Peptide based therapeutics offer the potential to target a much wider range of targets by inhibiting protein-protein interactions. The major drawbacks of peptide therapeutics include lack proteolytic stability and ill-defined secondary structures. These problems can be eloquently overcome by incorporation of unnatural amino acids into potential therapeutics to lower the rate of proteolytic cleavage and by creating cyclic peptides to lock the peptide into a conformation which lowers the entropic penalty of binding. Indeed, there are numerous examples of unnatural peptides exhibiting enhanced pharmacokinetics over their parent peptides. Taking in all these considerations, the ability to create libraries of random peptides containing unnatural amino acids is an appealing endeavor. Through a reconstituted cell-free translation system we demonstrate how an editing deficient valine tRNA synthetase (ValRS T222P) can be used for the incorporation of 13 unnatural amino acids into peptides including three cyclic beta amino acids and three alpha methyl amino acids. This system has the potential to be applied to the ligand discovery tool of mRNA display for identification of potent peptide inhibitors of disease relevant protein-protein interactions.