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New effective therapies are greatly needed for metastatic uveal melanoma, the most common ocular malignancy, which has a very poor prognosis with a median survival of <1 year. The melanocortin 1 receptor (MC1R) is expressed in 94% of uveal melanoma. MC1R is not expressed in normal tissues of concern for toxicity. We have previously reported a MC1R specific ligand (MC1RL) with high affinity and selectivity for MC1R. Ac-225 is a therapeutic radionuclide that emits 4 alpha particles in the decay chain. The aim of this study is to use MC1RL as a targeting scaffold for development of a radiopharmaceutical by conjugation of Ac-225 chelate.

The purification process of crude synthetic peptides as well as recombinant Insulin typically includes a reversed phase high performance liquid chromatography (HPLC) step. A single step process is most desired but a multiple step process is often necessary to achieve purity greater than 98% especially with long peptide and recombinant proteins like human Insulin. When a multistep process is used, the first steps are designed to remove most of the undesired components whereas the last step is developed to further “polish” the API to the desired purity level.

This presentation will cover the current state of the art for large scale preparative HPLC purification of synthetic peptide. The chemical and physical property of the purification packing material will be discussed as well as the mobile phase and critical process parameter needed to develop robust and scalable process. A revolutionary hybrid silica based stationary phase designed for the purification of peptide/proteins prone to aggregation will be highlighted and several purification case studies such as GLP-1 analogues and Insulin analogues will be presented.

Peptide-based research tools are a valuable asset in performing basic biological research. The availability of for consistent, high quality reagents is a vital element in the researcher’s toolbox in order to study areas such as receptor binding, and cell biology.

Total chemical synthesis offers the researcher a high degree of control in the design of standard and bespoke peptide-based research tools. The nature of solid phase peptide synthesis (SPPS) allows the incorporation of natural or unnatural modifications in a highly specific manner leading to reproducible manufacturing methods and product quality. Historically, SPPS methods limited the length of peptides that could be produced chemically to 40-60 amino acids, but modern methods have now pushed the boundary beyond 100 amino acids enabling small proteins to become legitimate targets for synthesis.

Almac has developed methods in SPPS in the development of relevant peptide / small protein targets such as chemokines (70-80 amino acids), ubiquitin reagents (76 amino acids), and histones (ca. 135 amino acids). Historically, recombinant methods of production have been able to cope with the sheer length of the sequences, but been nor been able to control the introduction of site-specific modifications, such as fluorophores, or post-translational modifications such as methylations or phosphorylations. Almac has perfected methods of synthesis of such challenging molecules to produce physiologically relevant reagents that previously were not obtainable.

In this paper, we describe the synthetic methods employed towards a range of highly complex protein-based research tools, and the advantages they bring to the research community.

Peptides are ideal drug candidates due to their inherent high potency, low toxicity, and their ability to effect a broad range of targets [1]. With several high revenue peptide drugs on the market and a full pipeline of potential candidates [2], the demand for a highly robust and effective synthetic method is of great importance [3].
Currently, peptide synthesis research and production both face similar challenges – a sluggish and wasteful workflow in desperate need for optimization. The typical conventional optimization steps usually take a shotgun approach: screen resins, screen different reagent excesses, screen activators, etc. This synthetic process necessitates tens or hundreds of reactions, all of which can often take weeks or months to complete while requiring a great deal of time, money, and resources.
To address the needs of the market, new cGMP methodology has been developed utilizing automation and microwave assisted heating. This work details mechanistic-based, innovative improvements to the chemical methodology of solid phase peptide synthesis, application of these improvements to high-throughput SPPS for personalized medicine via peptide vaccines [4,5] and large scale peptide production with cGMP considerations.
[1] D. J. Craik, D. P. Fairlie, S. Liras, D. Price. Chem Biol Drug Des, 2013, 81, 136.
[2] K. Fosgerau, T. Hoffman. Drug Discov Today, 2015, 20, 122.
[3] J. M. Collins, K. A. Porter, S. K. Singh, G. S. Vanier. Org. Lett. 2014, 16, 940.
[4] P. A. Ott et al. Nature, 2017, 547, 217.
[5] R. Takahashi et al. Breast Cancer Research, 2014,16, R70.

Facile synthesis of C‐terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid‐phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C‐terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.

Since Merrifield has developed solid-phase peptide synthesis (SPPS) method, polystyrene (PS) based resins have been utilized mostly in peptide synthesis. For efficient SPPS, we have developed several kinds of core-shell type resins, in which reactive functional groups are mainly located on the shell layer of the resins.The core-shell (CS) structure was designed to overcome the diffusion problem and for easy accessibility of reagents, and washings. Because of this distinctive structural feature, the CS type resins afforded high performance in peptide synthesis and even photolytic cleavage reactions.
These resins were prepared by suspension co-polymerization, two-step polymerization, cross-linking core backbone, partial hydrolysis, and biphasic functionalization methods. Recently, we have developed diffusion-controlled functionalization method to obtain CS type PEGylated PS resin. To evaluate the performance of the resins, various peptide sequences were synthesized, and the results were compared with those of non-CS type PS resin and other PEG-based resins. Those resins gave excellent performance in peptide synthesis as well as screening peptide ligands form peptide libraries. When properly designed, the PEGylated resin could also be used for the synthesis of oligonucleotide, and purification of therapeutic antibodies.

[1] Kim, H.; Cho, J. K.; Chung, W. J.; Lee, Y. S. Org. Lett. 2004, 6, 3273. [2] Lee, T. K.; Ryoo, S. J.; Byun, J. W.; Lee, S. M.; Lee, Y. S. J. Comb. Chem. 2005, 7, 170. [3] Lee, T. K.; Lee, S. M.; Ryoo, S. J.; Byun, J. W.; Lee, Y. S. Tetrahedron Lett. 2005, 46, 7135.
[4] Cho, H. J.; Lee, T. K.; Kim, J. W.; Lee, S. M.; Lee, Y. S. J. Org. Chem. 2012, 77, 9156. [5] Kim, J; Kim S.; Shin, D.S.; Lee, Y.S. J. Peptide Science, 2018, 24:e3061.

Early Development of a KISS1/GPR54 Receptor Agonist for Prostate Cancer

Yvonne Angella, Yan Wanga, Yun Wub, Wen Jieb, Chen Haixia b, Yanfen Tengb,
Meng Xiangminb, Krishna Kodukulac, and Robert Drakasc

aDepartment of Peptide Chemistry, ChemPartner SSF
bDepartment of Peptide Chemistry, ChemPartner Shanghai
cShangPharma Innovation, 280 Utah Avenue, Suite 250, SSF, CA 94080

Metastin/kisspeptin is a C-terminal amidated peptide of 54 amino acid residues in length isolated from human placental tissue. It is a ligand of the orphan G-protein-coupled receptor KISS1R, also called GPR54, which is expressed throughout the central nervous system and in a variety of endocrine and gonadal tissues. Kisspeptin plays a pivotal role in controlling gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin acts directly on the GnRH neurons to stimulate GnRH release.1 Compared to the full-length metastin protein, the N-terminally truncated peptide metastin(45–54) has 3–10 times higher receptor affinity and enhanced ability to increase intracellular calcium concentration, which is essential for activation of protein kinases involved in intracellular signaling in a number of pathways that affect reproduction and cell migration.2 KISS1R receptor agonists and antagonists have been explored to investigate the biology of targeting the KISS1 receptor. Metastin(45–54) analogues with higher agonist activity and improved metabolic stability have been reported in the literature, and shown to suppress plasma testosterone in male rats with continuous subcutaneous administration.3 Several KISS1R agonists have reached phase II in the clinic.4

Continuous subcutaneous administration of KISS1R agonists has been shown to induce a transient increase in plasma testosterone, followed by abrupt reduction of plasma testosterone to castrate levels within 3–7 days, which can be sustained throughout a 4-week dosing period. These suppressive effects are more rapid and profound than those induced by the GnRH agonist analog leuprolide.3 Hormonal therapy involving luteinizing hormone (LH)-releasing hormone agonists (LHRHs) is a widely used systemic treatment for prostate cancer and is recommended in the European Association of Urology guidelines on prostate cancer for use in patients with locally advanced and metastatic disease.5 We sought to design novel peptides with increased activity against KISS1R along with significantly enhanced plasma stability over previously reported KISS1R agonist compounds, and demonstrate in vivo efficacy of these novel peptides. The peptides were rationally designed based on Ala scanning and known SAR, and screened in vitro in a GPR54-NFAT-bla CHO-K1 cell-based primary assay, as well as a cell migration secondary assay used to test the peptides’ effect on migratory properties of cells stably expressing human KISS1R. Lead peptides selected from the in vitro results were screened for plasma stability and in vivo efficacy. The lead peptide, YA-156, demonstrated potent in vitro as well as in vivo efficacy in a subcutaneous, xenograft x1LnCAP BALB/c nude mouse prostate cancer model, excellent plasma stability, and a good in vitro ADME profile, aside from some unexpected receptor selectivity results.

Further studies are currently in progress to assess the in vivo effects of these novel KISS1R analogs. Chronic administration of kisspeptin analogs via a sustained release formulation may hold promise of a novel therapeutic approach for suppressing reproductive functions and hormone-related diseases, such as prostate cancer.

1. Kotani, M. et al., J. Biol. Chem., 2001, 276(37), 34631–34636.
2. Ohtaki, T. et al., Nature, 2001, 411, 613.
3. Matsui, H. et al., Eur. J. Pharmacol., 2014, 735, 77–85.
4. MacLean, D.B. et al., J. Clin. Endocrinol Metab., 2014, 99(8), E1445–E1453.
5. EAU Guidelines on prostate cancer, 2007, www.uroweb.org.

Yan Wang a, Yvonne Angell a, Yun Wu b, Yanfen Teng b, Meng Li b, Krishna Kodukula c and Robert Drakas c
a. Department of Peptide Chemistry, ChemPartner SSF;
b. ChemPartner Shanghai;
c. ShangPharma Innovation, 280 Utah Avenue, Suite 250, SSF, CA 94080

Chemerin is an immunomodulating factor secreted in many tissues including the spleen, lymph nodes, adipose tissue and epithelia. It was identified as a ligand of the Chemerin receptor (ChemR23 / CMKLR1), a chemokine like G protein-coupled receptor (GPCR) and induces chemotaxis in natural killer cells, macrophages, and immature dendritic cells. It is secreted as an inactive precursor, pro-chemerin, and is activated by proteolytic removal of amino acids from the c-terminus by proteases such as elastase, cathepsin G, or kallikrein 7. The nonapeptide, chemerin-9, derived from the C-terminus of the processed form of chemerin is also a potent agonist of CMKLR1.
Chemerin is involved in a variety of functions including cell differentiation and has been linked with diverse indications such as inflammation, psoriasis and obesity. However, its mechanism and detailed signaling pathway remain unclear. Also, chemerin and chemerin-9 are rapidly degraded and inactivated in plasma, which has limited their use in in vivo experiments and as potential therapies. There is a need to develop stable and selective molecular tools to accelerate pharmacological analysis of Chemerin–CMKLR1 interactions, better define signaling pathways, and screen in vivo to validate CMKLR1 as a potential therapeutic target.
We carried out multiple peptide modification strategies and tested more than 230 peptides in an SAR study, using human CMKLR1 over-expressed in U2OS cells as our primary assay. A number of potent analogs were identified, among them, two peptides were chosen as lead candidates for further studies. Both analogs showed good metabolic stability and no toxicity. They also had excellent GPCR selectivity during GPCR panel screening at 10 µM concentration.
Thus, these analogs have emerged as useful tools for further interrogation of chemerin’s biological role and as potential lead structures for continued development.

Measuring cell permeability of peptides is critical for understanding and optimizing activity of ligands with intracellular targets and assessing potential for oral bioavailability. While small molecule permeability has been extensively studied, methods for assessing peptide permeability are often lower throughput, target-specific, or less robust. Herein we describe the Nanoclick assay, a high-throughput and target agnostic approach for assessing the permeability of azide-containing peptides. We present comparisons of Nanoclick and cell-based on-target activity, and cell-free reactivity data used to better understand cycloaddition kinetics.

Increased lifetime of RPC resins in insulin production by clean-up using WorkBeads 40S

Purification of recombinant insulin requires very high purity, often achieved by high-resolution reversed phase chromatography (RPC) based on silica. Impurities from the feed often cause fouling of the silica resins that is difficult to remove since the option of cleaning-in-place using sodium hydroxide is limited. These issues result in shortened lifetime of the RPC columns. We present here that an introduction of an ion exchange chromatography step before RPC removes the bulk of impurities from the feed which significantly will increase the lifetime of the RPC column.
By introducing a capture step, using WorkBeads™ 40S (a cation exchanger), a standard purification process involving a combination of two RPC steps with two different buffer systems (to obtain complementary selectivity) was improved.
WorkBeads 40S is an agarose based resin with sulfonate ligands which is stable in high concentration of sodium hydroxide thus allowing efficient cleaning-in-place. A feed containing 72.5% pure human insulin precursor (Met-Lys-Human insulin) was applied to the WorkBeads 40S column, followed by the two RPC steps. Each step was optimized to give a yield of 90% while maximizing the purity after each step. The amount of impurities in the applied feed on the first RPC column could be reduced from 27.5% to 12%. This improvement significantly reduces the fouling of the first RPC column and prolongs its lifetime. The final purity for the human insulin precursor was > 99%. A comparison was also done using Capto™ SP ImpRes cation exchange resin in the capture step which resulted in a less pure target protein compared to using WorkBeads 40S.