ABSTRACT:Obesity is a global health concern due to the disturbing rise in prevalence and extent. With cardiovascular diseases being the lead cause of mortality in patients there is a substantial unmet medical need to develop novel drugs targeting not only obesity, but also obesity associated cardiovascular diseases such as hypertension and atherosclerosis. More We hypothesize that combining the cardioprotective effects of adrenomedullin with the anti-obesity and glucoregulatory properties of amylin could be an effective treatment strategy. Here we show novel unimolecular peptide agonists with dual activity at both the amylin and adrenomedullin receptors. Using SPPS, a library of hybrid peptides was rationally designed by substituting amino acids essential for amylin activity into the native adrenomedullin sequence. The pharmacokinetic profile of the analogues was optimized by N-terminal lipidation with a C20 diacid. The functional activities of the analogues were assessed using a cAMP accumulation assay in cells overexpressing the human amylin receptor subtype 3 (hAMY3-R) or human adrenomedullin receptor subtype 1 (hAM1-R). All peptides were measured to be full agonists on both receptors. The hAMY3R potencies were equivalent to human amylin while potencies on the hAM1-R showed a range from equipotent to 100-fold less potent as compared to human adrenomedullin. Ongoing studies are aimed at addressing the efficacy and potency in rodent models of metabolic diseases. In summary we have designed, synthesized, and evaluated novel hybrid amylin-adrenomedullin peptide agonists with dual activity at the hAMY3R and hAM1-R for the potential treatment of obesity patients with high cardiovascular risk.
ABSTRACT:Peptide YY3-36 (PYY3-36) is an endogenous ligand of the neuropeptide Y2 receptor (Y2R), through which it acts to reduce food intake. Accordingly, PYY3-36 analogues are interesting as potential anti-obesity pharmaceuticals. However, native PYY3-36 is rapidly cleared from circulation, and half-life extension is thus a prerequisite for PYY3-36 based More pharmaceuticals. This is typically achieved by covalent attachment of PYY3-36 to macromolecules (e.g. PEG) or through lipidation which promotes non-covalent interactions to albumin. Many peptide drugs, like PYY3-36, require binding to a specific G protein coupled receptor (GPCR) to exert their effect. GPCRs are desensitized and internalized following intracellular binding of β-arrestins, which bind to the ligand-activated conformation of the receptor.
Beck-Sickinger and co-workers recently reported how PEGylation and lipidation differentially directs arrestin 3 (Arr3) recruitment and receptor internalization of obinepitide, another peptide of the neuropeptide Y receptor system. Hence, the half-life extender selected for PYY3-36 therapeutics may alter its efficacy. Accordingly, we aimed to investigate how three commonly applied half-life extenders, PEGylation (PEG20), hexadecanoic acid (C16), and octadecanedioic acid (C18-acid), directs Y2R-mediated internalization of PYY3-36. Here we report how PEGylation leads to a G protein bias and reduced Y2R internalization. We further report how lipidation with both C16 and C18-acid did not bias Y2R signalling, whereas only C16 increased Y2R internalization. Finally, we demonstrate how binding kinetics underlay some of these differences.
1 Rajagopal S et al. Nat. Rev. Drug Discov. 2010; 9: 373–386
2 Mäde V, et al. Angew. Chemie - Int. Ed. 2014; 53: 10067–10071
Poster ID# 4795
PEDF peptides internalize a receptor, inhibit retinal disease and cancer, while blocking amyloid neurotoxicity
ABSTRACT:Purpose: Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic and tumor suppressive 50kDa protein, decreased in neovascular retinopathies, and aggressive cancers. PEDF protective activity has been localized to a 34-mer surface peptide, then 25 and 18aa fragments therein. We sought smaller practical fragments, able to be modified as More prodrugs to nanocarriers, then attempted to evaluate their clinical potential in cell and animal models, and to identify their target receptor.
Methods: Truncation of the 18-25 mer fragments led to anti-angiogenic 8-10aa peptides screened by endothelial cell (EC) apoptosis after VEGF activation. Further changes identified N-terminal dicarboxylic acid (DCA) modified octa- and nonapeptides, where internal Asp to Asn residue substitution, optimized apoptotic response. Two potent small peptides (8-mer, 9-mer) by intravitreal (IVT) delivery, were tested in mouse models of choroidal neovascularization (CNV) and of ischemic retinopathy (OIR). The same peptides and cyanine dye-tagged analogs were tested against ovarian, prostate and glioma cells, also NS-1 neurons, examining apoptosis and endocytosis. The peptides were tested in tumor models and against amyloid cytotoxicity. Cell uptake of dye was examined after specific cell exposure to antibodies and free peptides.
Results: A single IVT injection of 2-4 nmol of either peptide resulted in a significant decrease in mouse CNV and in neonatal mouse OIR, with no signs of toxicity. Eye drop delivery (8mer) was effective in CNV and a prodrug nanoparticle conjugate (9mer) extended CNV protection. Peptides induced tumor cell apoptosis in both ovarian cancer in a glioma stem cells and inhibited ovarian cancer orthotopic tumor growth with QD systemic injection. They protected neural cells from apoptosis induction by amyloid peptide a,b 1-42. Brief exposure to PEDF, free peptide, and anti 67LR exposure block cyanine-peptide endocytosis in 3 cell types.
Conclusions: A small active pharmacophore was defined within the PEDF sequence (Y-D-L-Y-R-V). Optimization around ..YNLYR.. improves >100-fold on the laminin beta1 motif, YIGSR, which weakly binds non-integrin laminin receptor, 67LR. The latter plays harmful roles in angiogenesis, tumor malignancy, neuronal amyloid uptake and microbial/viral cell entry. New modifications (adipic-X-Y-N-L-Y-R-V…) yielded practical, synthetic peptides that can mitigate retinopathy and cancer growth, and protect neurons, via cell surface removal of 67LR.
Poster ID# 4830
Design, Synthesis and Activity of Biomimetic Macrocyclic Inhibitors of Human Cathepsin D
ABSTRACT:Human cathepsin D (CatD) is a pepsin-family aspartic protease, which is involved in many physiological and pathological processes in human. It is overexpressed in breast cancer cells and associated with tumor progression and metastasis. CatD inhibitors were proposed as potential cancer therapeutics and many compounds have been synthesized.
In this More study, we report the development of macrocyclic inhibitors inspired by natural inhibitor of CatD, Pepstatin A. The cyclic scaffold was designed to mimic spatial conformation of the minimal pseudo-dipeptide binding motif, while removing unnecessary amino acids and replacing them with a suitable bridge connecting P2 and P3´ positions.
Reported compounds are composed of three sub-units linked together. The first sub-unit (2-hydroxy-3-amino acid) contains the transition state isostere (hydroxy group), which interacts with the active site aspartates. The second sub-unit contains R2 substituent filling the S2’ sub-site, while the third one is a linker connecting the first two units and interacting with the large hydrophobic S2-S3’ site of the enzyme binding cleft. Library of over 30 compounds was employed for scaffold optimization. Synthesis and inhibitory potency of these compounds with variable size of the bridge and various side chains in P1 and P2´ positions will be reported.
Poster ID# 4868
PRODRUGS OF 6-DIAZO-5-OXO-L-NORLEUCINE (DON) WITH ENHANCED CSF DELIVERY IN MONKEYS AS A POTENTIAL TREATMENT FOR GLIOBLASTOMA
ABSTRACT:6-Diazo-5-oxo-L-norleucine (DON, 1) is a non-standard amino acid, which was originally isolated from Streptomyces bacteria found in Peruvian soil in 1956. DON is one of very few naturally occurring diazoketones and it was characterized by Dion. It has shown robust anticancer efficacy in preclinical and clinical studies, but development was halted More by significant systemic toxicity. To enhance DON’s therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Several prodrugs were prepared and the most stable compound 2 was tested in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON.
Poster ID# 4880
Tumor-targeted delivery of 6-Diazo-5-oxo-L-norleucine(DON) using substituted acetylated lysine prodrugs
ABSTRACT:6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy, yet its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal tissues which are highly glutamine-dependent. DON acts as an irreversible inhibitor of many glutamine utilizing enzymes critical More for the synthesis of nucleic acids/ proteins and the generation of α-ketoglutarate for energy metabolism. The anticancer and autoimmune activities of DON has been shown repeatedly in both preclinical and clinical studies.
Herein we describe the synthesis of a series of tumor-targeted DON prodrugs that were designed to circulate intact and inert in plasma and be cleaved to DON preferentially in tumor cells. Our best prodrug 1 showed stability in plasma, liver and intestinal homogenates, yet was readily cleaved to DON in tumor cells. When directly compared to DON, prodrug 1 exhibited a 27-fold enhanced tumor cell-to-plasma ratio.
Poster ID# 4897
Prodrugs of 6-Diazo-5-oxo-L-norleucine: Strategy for Delivery in Brain and Potencial Treatment of Lymphoma
ABSTRACT:6-diazo-5-oxo-L-norleucine (DON), a non-natural amino acid with structural similarity to glutamine, was first isolated from Streptomyces bacteria in the 1950s. It has shown promissing antitumor activity in preclinical and several clinical studies. DON acts as an irreversible inhibitor of many glutamine utilizing enzymes critical for the synthesis More of nucleic acids, proteins and the generation of α-ketoglutarate for energy metabolism. However, its high toxicity leading to gastrointestinal side effects prevented its further development. We hypothesized that a novel cell-directed prodrug of DON which could deliver the drug selectively to cells and would permit significant dose reduction, greatly alleviating the GI adverse events.
Herein we report the design, synthesis, and evaluation of several novel DON prodrugs targeted to Peripheral Blood Mononuclear Cells (PBMC’s). Using whole blood from mouse, pig, dog, monkey and human, we found that several of the new prodrugs selectively delivered DON into PBMC‘s versus plasma by >10-fold. These findings open an opportunity to develop therapeutics active at lower dose circumventing dose limiting toxicities.
 Rais, R.; Jančařík, A.; Tenora, L.; Nedelcovych, M.; Alt, J.; Englert, J.; Rojas, C.; Le, A.; Elgogary, A.; Tan, J.; Monincová, L.; Pate, K.; Adams, R.; Ferraris, D.; Powell, J.; Majer, P.; Slusher, B. S. J. Med. Chem. 2016, 59, 8621.
 Nedelcovych, M.; Tenora, L.; Kim, B.-H.; Kelschenbach, J.; Chao, W.; Hadas, E.; Jančařík, A.; Prchalová, E.; Zimmermann, S. C.; Gadiano, A.; Garrett, C.; Furtmüller, G.; Oh, B.; Brandacher,G.; Alt, J.; Majer, P.; Volsky, D.J.; Rais, R.; Slusher, B.S. J. Med. Chem. 2017, 60, 7186.
Poster ID# 4898
Rapid Discovery of Illuminating Peptides for Instant Detection of Opioids in Blood and Body Fluids
ABSTRACT:The United States is currently experiencing an opioid crisis, with more than 47,000 deaths because of overdose in 2017. Current approaches for opioid identification and quantification in body fluids include immunoassays and chromatographic methods (e.g., LC-MS, GC-MS) which require expensive instrumentation and extensive sample preparation. Our aim More is to develop a portable point-of-care device that can be used for instant detection of opioids in body fluids. Here, we report the development of a morphine-sensitive fluorescence-based sensor chip to sensitively detect morphine in blood using a homogenous immunoassay without any washing steps. Morphine-sensitive illuminating peptides were identified by a high throughput OBOC combinatorial peptide library approach. The OBOC libraries containing a large number of random peptides with a molecular rotor dye were screened for fluorescent activation under a confocal microscope as shown in figure 1. Using this novel three-step fluorescent screening assay, we were able to identify peptide-beads that fluoresce in the presence of anti-morphine antibody, but lost fluorescence when free morphine was present. Positive beads were then decoded using automatic Edman microsequencing, and the morphine-sensitive illuminating peptides were synthesized in soluble form, functionalized with an azido group, and immobilized onto microfabricated PEG-array spots on a glass slide. The sensor chip was then evaluated for detection of morphine in plasma. We have demonstrated that this proof-of-concept platform can be used to develop fluorescence-based sensors against morphine. More importantly, this technology can also be applied to the discovery of other novel illuminating peptidic sensors for detection of illicit drugs and cancer biomarkers in body fluids.
Poster ID# 4903
Cancer-Specific Intracellular Delivery of Therapeutic Antibodies Against RAS
ABSTRACT:Antibodies and peptides directed against the switch regions of RAS can disrupt interactions with its downstream effector proteins, blocking downstream pro-tumorigenic signaling. However, these biologic therapies are unable to reach the intracellular environment. We have overcome this problem using peptidic molecular guidance system (MGSs) that have More the unique ability to bind specifically to epithelial-derived cancer cells, and upon binding, trigger rapid internalization and trafficking to a specific subcellular location. Using an unbiased phage display approach, we have identified a series of cancer-specific MGSs that deliver cargo to discrete subcellular locations. Chemical optimization results in MGSs with 1-10 nM affinity for their cellular target, serum stability >48 hours, and 50-1000-fold specificity for cancer cells compared to normal control cells. The rate of MGS internalization is rapid with t1/2 of 10-30 minutes, achieving intracellular concentrations up to 1.5 µM. Using these MGSs as delivery agents, we have delivered a function blocking RAS antibody (Y13-259), resulting in a reduction of p-ERK and apoptosis in KRAS mutant cell lines. The effect is MGS dependent, and confocal microscopy indicates co-localization of KRAS and the antibody indicating target engagement. The MGS-Antibody homes to tumors in an animal model resulting in significant suppression in tumor growth compared to untreated animals. Free MAb does not impact tumor growth. Using MGSs to delivery MAb for intracellular targets is paradigm change for immunotherapy and represents a new approach for controlling intracellular protein-protein interactions. Combination of these cancer-specific delivery agents with effectors of RAS signaling, such as antibodies and other biologics, has therapeutic potential for KRAS-driven cancers.
Poster ID# 4905
Cancer-Specific Peptidic Ligands Enhance Delivery of Anti-Sense Oligonucleotides
ABSTRACT:Therapeutic applications of antisense oligonucleotides (ASOs) will be expanded by methods that direct ASO uptake into targeted cell types and deliver the ASO to desired subcellular locations. With this objective, we have applied novel peptidic molecular guidance systems (MGSs) to facilitate cell-specific ASO delivery. These MGSs have the unique More ability to bind to specific epithelial-derived cancer cell types and, upon binding, trigger rapid internalization and trafficking to specific subcellular locations. The chemically optimized MGSs have 1-10 nM affinities for their cellular targets, serum stability greater than 48 hours, and 50-1000-fold specificity for targeted cancer cells over normal control cells. Target cells internalize these peptidic ligands rapidly, reaching intracellular concentrations up to 1.5 µM with t[1/2] of 10-30 minutes. Conjugates of stabilized ASOs with these MGSs show robust, temperature-dependent uptake into human lung cancer cells, observed with flow cytometry and fluorescence microscopy. Cell-type specificity for conjugate internalization confirms MGS specificity. Furthermore, MGS-mediated delivery enhances ASO potency: conjugation of an MGS to a Generation 2.5 ASO targeting MALAT-1 increases ASO potency in the targeted cancer cells by up to seven-fold compared to the unconjugated ASO. Conversely, antisense activity of the conjugated ASO is markedly reduced in cells not targeted by the MGS. These results demonstrate a strategy for cell-specific delivery of ASOs as the cargo of engineered cell-targeting peptides.
Poster ID# 4908
A Novel Peptide-Guided Immunotherapy for Treatment of Lung and Breast Cancer
ABSTRACT:Cancer Immunotherapies designed to generate a cell-mediated immune response against tumors are emerging as frontline treatment options for cancer; however, concerns regarding efficacy, safety and cost efficacy have limited the use of these treatments. To address these weaknesses, we have developed a novel immunotherapy that utilizes a More tumor-targeting peptide capable of delivering previously encountered antigenic peptides specifically to cancer cells and facilitating their presentation through the MHC class I pathway. Our therapy utilizes a modular synthetic nanoparticle delivery system comprising of three components: a neutral stealth liposome, encapsulated synthetic immunogenic HLA class I restricted peptides derived from measles virus (MV), and a tumor-targeting peptide on the external surface of the liposome. The targeting peptide results in accumulation of the liposomes specifically inside cancer cells and facilitates presentation of the MV-derived immunogenic peptides in HLA class I molecules. We refer to this system as TALL (Targeted Antigen Loaded Liposomes). Therefore, TALL can generate a secondary immune response specifically against the targeted tumor cells in a patient who has been previously vaccinated against or infected by MV. In short, we are attempting to trick the immune system into responding as though the cancer cell is infected with MV without the use of a viral particle. The tumor targeting peptide has undergone chemical optimization to improve cell specificity, affinity, solubility and biodistribution. The mechanism of cellular internalization of the liposome mediated by the targeting peptide has been elucidated. We have demonstrated a significant reduction in subcutaneous tumor growth using P-TALL in aggressive LLC1 and 4T1 murine models with no gross toxicity.
Poster ID# 4911
Chemoselective Macrocyclization of Tyrosine and Tryptophan Containing Peptides
ABSTRACT:Peptide-based natural products have long served as inspiration in the development of new therapeutics for the treatment of human disease. Therapeutic peptides commonly exhibit effective protein-target binding activity at low concentrations as a result of high selectivity. However, the value of peptide therapeutics is often overshadowed by a marked More susceptibility to proteolytic degradation and subsequent failure to reach a validated target in vivo. The modification of bioactive linear peptides by macrocyclization has shown to be a promising strategy for addressing this problem. Drawing inspiration from Nature’s wide collection of non-ribosomal peptides, specifically those comprising electron-rich aromatic moieties, we aim to augment current methodology by developing new chemical strategies for the synthesis of cyclic and stabilized peptides by leveraging the inherent nucleophilicity of Tyr and Trp residues. Due to their remarkable reactivity toward various aromatic nuclei such as phenols (Tyr) and indoles (Trp), we have identified N-4-substituted azo reagents, namely, 1,2,4-triazoline-3,5-diones (TADs), as chemoselective electrophiles for the construction of macrocyclic peptidomimetic scaffolds. Upon synthesizing an N4-substituted 1,2,4-triazolidine-3,5-dione (aka urazole) at the N-terminus of a solid-supported peptide, the urazole moiety is chemoselectively oxidized under mild conditions to generate a TAD derivative in situ. The subsequent reaction of this electrophilic TAD moiety with the sidechain aromatic nuclei of internally or terminally located Tyr or Trp residues should result in the chemoselective formation of peptidic macrocycles. This poster will give an overview of recent progress concerning the development of this method, as well as the successful synthesis of macrocyclic peptides wherein the TAD moiety has been trapped by the phenolic sidechain of Tyr. Results regarding LC-MS, 1-D NMR, and 2-D NMR experiments will be discussed. Furthermore, preliminary evidence which supports the trapping of the electrophilic TAD by the sidechain of Trp will be presented.
Poster ID# 4912
Efficient methods for rapid development of therapeutic peptides with higher structural complexity
ABSTRACT:Method development for the manufacture of therapeutic peptides remains a vital piece of the commercial peptide production process. An optimized solid-phase synthesis protocol can be challenging to develop. Recent advances in peptide therapeutics focus on greater structural complexity making peptides that are more physiologically stable products More with increased target specificity and membrane permeability . Peptides are also key players in the personalized medicine sector, which relies on faster peptide synthesis protocols and reduced processing times to reach patients effectively.
Here we show the complete parallel synthesis, from swelling to cleavage, of several biologically relevant peptides under different conditions, including increased temperatures (50-90°C), on an automated peptide synthesizer in high purity. In some examples, real-time UV deprotection monitoring has been used to assist in the synthesis optimization process. Optimization has also allowed a reduction in total synthesis times for preparation of the pure peptides.
 S. H. Joo, “Cyclic peptides as therapeutic agents and biochemical tools,” Biomol. Ther., 20, 1,19-26, 2012.
Poster ID# 4916
Total Chemical Synthesis of Human C5a Protein with Assistance of Pullover Peptides
ABSTRACT:Tong ZZ, Jacobsen MT*
The scale of total chemical synthesis of proteins has increased significantly over the last 25 years. However, one major challenge in synthesizing proteins is the tedious HPLC purification of synthetic intermediates, which becomes more difficult as the target length increases. Often, one difficult peptide segment complicates More the HPLC purification of a target, ultimately thwarting the project. In this presentation, we provide a solution to this chromatographic dilemma. We demonstrate how the judicious application of optimized NCL (native chemical ligation) reactants, termed pullover peptides, can simplify the purification of challenging NCL reactions. We rationally designed two model peptides, ThiP (thioester pullover) and CysP (Cys pullover), and then evaluated their chromatographic effects on model peptides. Pullover peptides are added to completed NCL reactions to cause HPLC shifts in unreacted (non-ligated) peptides.
Using a ThiP pullover peptide, we streamlined the synthesis of the 74-amino acid human C5a protein. C5a is a pro-inflammatory cytokine, especially associated with acute lung injury, which has been intractable to specific antibody therapies. Our initial synthesis of C5a was complicated by ligation at a poorly-reactive Thr thioester and co-elution of reactant and product during final HPLC purification. Using optimized conditions with a pullover peptide, we achieved synthesis and purification of multiple C5a proteins including wild-type, mutant, Biotin-labeled, and Fluorescent-labeled versions. All proteins were then refolded and characterized with respect to recombinantly-expressed protein: the wild-type and labeled proteins behaved indistinguishably from recombinant protein, while the designed mutant was functionally inactive. Pullover peptides are straightforward to prepare and implement, and they can be used generally to simplify HPLC purification in total chemical protein synthesis.
Poster ID# 4959
From Helping Hands to Helping Handcuffs: dimedone esters in solid-phase peptide synthesis
ABSTRACT:Moore A*, Chugg A, Kawamoto EM, Jacobsen MT
The reaction of primary amines with acetyl dimedones has been used to create the well-established Dde (N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) protecting group in solid-phase peptide synthesis (SPPS). We recently utilized this reaction to introduce solubilizing groups (“Helping Hands”) onto Lys More side chains of difficult peptides with the Fmoc-Ddae-OH linker. Helping Hands can be cleaved with a nucleophile, usually hydrazine, to restore the primary amine. In the first part of this presentation, we present advice on the scope of acetyl dimedone incorporation and removal kinetics in a diversity of SPPS contexts.
Acetyl dimedones and its analogs are generally prepared in a one-pot, two-step reaction involving a carboxylic acid and dimedone. During the course of developing new acetyl dimedone derivatives, we recognized that the key synthetic intermediate, dimedone ester, is an easy-to-prepare, useful, and highly-solubilizing group for peptide synthesis. In contrast to the acetyl dimedone, the dimedone ester (“Helping Handcuff”) creates a non-cleavable amide bond with peptide amines. In the second part of this presentation, we showcase the versatility of dimedone esters; in particular, we note their significant solubilizing benefit under SPPS conditions (e.g., 10-30x greater solubility compared to the corresponding NHS ester). Finally, we demonstrate the overall utility of both Fmoc and biotin-dimedone esters for general peptide synthesis.
Poster ID# 4960
A novel small peptide-based therapy for advanced prostate cancers
ABSTRACT:Prostate cancers at the lethal stage of castration resistance have no means to cure in the clinic. We invented a small peptide approach to trigger a client protein-specific degrading signal for destroying the androgen receptor (AR) protein, the most critical driver in newly developed and post-Abiraterone/ Enzalutamide castration-resistant prostate More cancers. The small peptides were derived from the N-terminal segment of the AR protein and were conjugated with a plasma membrane penetrating peptide for prostate cancer-specific uptake. Treatment of AR-positive prostate cancer cells with the small peptides dramatically reduced AR protein levels without affecting the mRNA levels. The AR reduction was not dependent on the ubiquitin-proteasome pathway since the co-treatment of prostate cancer cells with the small peptides with the proteasome inhibitor MG132 did not prevent AR protein reduction. Intra-tumoral injection of the peptides in prostate cancer cell-derived xenografts in nude mice also resulted in suppression of tumor growth. In conclusion, our data suggest that this small peptide approach is feasible to be developed as a novel therapy for advanced prostate cancers.
Poster ID# 4964
Novel Insulin-oligosaccharide Conjugates for Diabetes Treatment with Low Hypoglycemia Risk
ABSTRACT:Insulin and its analogs used in the diabetes treatment are often insufficient due to the inability of these exogenous insulins to function in response to the varying glucose concentration and resulting hypoglycemia. A novel approach to improve such narrow therapeutic index by conjugation of a cluster of sugars, e.g., D-mannose and L-fucose, to More insulin, has been explored to potentially offer glucose responsive insulins with low hypoglycemia risk. The cluster of sugar moieties, acting as substrate of endogenous mannose receptor (MR), have been shown to affect the pharmacokinetic properties of their corresponding insulin conjugates in a way that is sensitive to the endogenous glucose concentration, rendering these insulin conjugates low risk of hypoglycemia. This presentation will cover our effort in the design and synthesis of insulin oligosaccharide conjugates (IOCs) that showed efficacy in clinically relevant minipig models with /without the presence of α -methyl mannose, a glucose surrogate; and our effort of developing a potent follow-up candidate to our first clinical candidate MK-2640.
Poster ID# 4966
Evolvable Minimal Tertiary Mimics of Protein-Protein Interactions
ABSTRACT:Rationally designed protein domain mimics are based on secondary and tertiary structure epitopes observed at protien-protein interfaces. Successful development relies on interplay between good quality crystal and computational data, with iterative synthesis of scaffolds displaying key residues involved in binding. These direct mimics are limited in More their binding affinity by the functional transience and low affinity of the native complex and require significant optimisation. We seek to translate an optimised tertiary mimic scaffold, a crosslinked helical dimer, to a phage screening platform that would allow access to diverse libraries of conformationally defined interfaces. The libraries could then be refined against a target of interest and amplified for further rounds of selection, deviating from the need to define the target epitope by structural analysis. With our developed library, we screened against VEGF, identifying 8 unique sequences which have been shown to bind by ELISA, and in vitro binding studies using fluorescence polarisation are underway.
Poster ID# 4978
High Throughput Discovery of Novel Peptides with Biological Function
ABSTRACT:Peptides are highly active biological molecules that, with advances in stability and delivery, present huge potential as drug candidates. With an increased interest in the use of peptides as therapeutics comes the need for strategies to allow for the discovery of novel hit candidates, in a high throughput manner, from highly complex peptide More libraries.
Current technologies for the discovery of novel peptide therapeutics include phage, yeast, ribosomal and mRNA display but these all hold their own limitations. Phage and yeast display are sensitive but a reliance upon a biological host can result in replication bias and interference from native host proteins. These systems are also restricted to the use of natural amino acids. In vitro technologies based on mRNA and ribosomal display have the ability to incorporate unnatural amino acids but are limited by small peptide copy number leading to a reduced detection of low affinity binders. Moreover, none of the current peptide display technologies can effectively address cell surface targets for the discovery of functional peptides.
The ORBIT in vitro display technology uses beads to present randomised peptide sequences and the DNA which encodes them, thereby linking genotype to phenotype, and combines the advantages of in vitro display, such as highly diverse libraries and a cell-free environment, with the high sensitivity of in vivo technologies. The ORBIT display platform presents thousands of identical peptide copies per bead allowing for the discovery of low affinity binders and allows for the incorporation of unnatural amino acids and the generation of cyclic peptides which can increase chemical diversity and improve stability, specificity and affinity. In addition to screening for binding peptides, the ORBIT platform can be adapted to screen whole cell surfaces for peptides which elicit functional cellular responses.
In summary, the ORBIT peptide display platform offers peptide drug screening with high diversity, sensitivity and plasticity, and has the potential to discover novel, selective and functional peptide therapeutic leads with a broad target coverage, including cell surface targets, for a wide range of diseases.
Poster ID# 4981
Innovative Preclinical Assessment Tools for Safety and Efficacy of Protein and Peptide Therapeutics … Of Peptides and P-ANDAS
ABSTRACT:The peptide drug market has rapidly expanded and is expected to generate in excess of $50 billion for manufacturers. While synthetic peptide synthesis is a cost-effective approach for producing these drugs, regulatory agencies are concerned that impurities resulting from the manufacturing process could introduce an unwanted immune response. More Impurities can result from changes in the sequences due to deletions, insertions, substitutions, modifications and other impurities related to the synthetic production.
The FDA recently released a new draft guidance defining the equivalence of a rDNA peptide product and a synthetic peptide product enabling generic manufacturers of peptide drugs to file an Abbreviated New Drug Application (ANDA) for synthetic peptide drug products that refer to listed drugs of rDNA origin. Since the processes for manufacturing the generic and reference drug (RLD) are not equivalent, peptide drugs can be associated with impurities. The FDA draft guidance requires manufacturers to prove that synthetic peptide products do not contain impurities that have an increased affinity for major histocompatibility complexes and potential for engaging immune response, which may drive anti-drug antibody development.
Here we describe our PANDA assay that utilizes a combination of in silico assessment tools and in vitro assays to predict and validate the effect of peptide impurities on the immunogenicity of synthetic peptide drug products. In addition, we have developed a novel in-silico tool, the “What-if machine”, that mimics the process of synthetic peptide manufacturing and predicts the impact of impurities at any position in the sequence of a peptide drug.
Step 1. Immunoinformatics assessment: The potential of the DS to stimulate a T cell response can be rapidly assessed computationally using T cell epitope mapping algorithms. We use EpiMatrix for this purpose and focus on HLA DR (Class II) HLA binding predictions. In a typical DS analysis, the EpiMatrix algorithm is used to screen the primary amino acid sequence of the DS and its impurities, for the presence of HLA DR ligands, which can be considered putative T cell epitopes. Discriminating between potential inflammatory “T effector” epitopes and regulatory “T reg” epitopes is performed with a second algorithm, known as JanusMatrix. The latter algorithm identifies putative Treg epitopes, defined as HLA/epitope complexes that present a human-like outer contour (TCR face). HLA/epitope complexes that do not present an outer contour (TCR face) that is ‘human-like’ are more likely to drive effector T cell response. Following assessment of T cell epitope phenotype, the next step is to combine the scores for effector and regulatory T cell epitope content, providing an overall assessment of immunogenic potential of the DS and impurities. The resulting Treg adjusted EpiMatrix scores are highly correlated with immune responses in vivo.
Step 2: In vitro - HLA binding: The DS, its impurities, and peptides representing predicted T cell epitopes can be evaluated for binding to human HLA in assays that measure binding affinity in dose-ranging studies, in vitro. HLA binding is used to confirm in the in silico analysis and inform the design of in vitro assays (see step 3).
Step 3: In vitro assay – Teff Assay: Measurement of de novo T cell response: Cell culture protocols have been developed to emulate in vivo conditions that support differentiation of naïve T cells to effector T cells by antigen stimulation with biologics or their constituent T cell epitopes. In vitro stimulations using the biologic drug and human peripheral blood cells (PBMC) allow for natural antigen processing of the DS and other product components including impurities. In vitro assays using predicted epitopes derived from the DS product impurities provide information about the ability of these defined sequences to drive a T cell response using human T cells. At the end of the culture period, T cell phenotype and/or function are characterized in assays that measure the magnitude and quality of effector T cells that have potential to drive ADA development. Treg Assay: Co-incubation of the putative Treg epitopes with known T effector epitopes (such as Tetanus Toxoid-derived T effector epitopes) allows the assessment of bystander suppression. Bystander suppression is a feature of Treg epitopes.
In summary, in silico and in vitro assessment of novel T effector (inflammatory) and Treg (suppressive) epitopes is necessary to best evaluate the impact of novel impurities on the immunogenicity risk in humans. In addition to the standardized tools described above, we will describe novel in silico methods that have been developed to anticipate well-known synthetic peptide impurities (The What If Machine). A case study of Taspoglutide will be provided to illustrate these well-established (and more recently developed) Immunogenicity Risk Assessment methods.
Poster ID# 4982
Predicting Immunogenicity of Peptide Drugs and their Impurities using in Silico tools: Taspoglutide case study.
ABSTRACT:The peptide drug market is expected to generate $50B in revenue for companies but the FDA is concerned about the number of impurities that may be introduced in the synthetic process. Advances in chemical peptide synthesis have allowed the production of synthetic peptide drugs to become easier and more cost effective. However, the peptide More manufacturing process can result in synthesis-related impurities that can introduce immunogenic epitopes within the amino acid sequence of the peptide resulting in an unexpected and undesired immune response against the drug. For instance, during the process of solid phase synthesis, impurities may arise as the result of incomplete coupling and side reactions. Several classes of impurities related to manufacturing and degradation pathways exist and include: amino acid insertions and deletions, truncations, isomerization, and side chain modifications. Any of these impurities can affect the safety and efficacy of these drugs and it is therefore important to assess any impurities that may be present after synthesis.
T cell- (thymus-) dependent (Td) responses play a critical role in the development of antibody responses to biologic therapeutics. Td responses, by definition, are contingent upon T cell recognition of therapeutic peptide-derived T cell epitopes through the basic processes of antigen processing and presentation. A fundamental requirement for peptides to be considered as T cell epitopes is that they bind to human leukocyte antigen (HLA) molecules. The HLA-peptide interaction involves binding of specific amino acid side-chains to pockets in the HLA molecule binding ‘groove’, an interaction that is well-characterized for many HLA. Briefly, the requirements for HLA binding can be codified in ‘motifs’ or patterns. Based on these characterizations, pattern-matching algorithms, such as the EpiMatrix algorithm, have been developed to screen amino acid sequences for peptides that will bind HLA. EpiMatrix can be used to screen both the drug API sequence and its known peptide-related impurities. When peptide-related impurities are unknown, modifications at each amino acid position in the peptide can be performed in silico, their immunogenicity risk can be predicted, and they can be assigned an impurity risk score. The “What if Machine” (WhIM), performs all possible changes to the natural amino acid sequence of the drug substance and measures their impact on the epitope content of the peptide.
Here we present a retrospective case study of Taspoglutide – a GLP-1 agonist that was under investigation for the treatment of type 2 diabetes, but development was halted during phase III clinical trials due to serious hypersensitivity reactions and GI intolerance. It is suspected that the cause of the observed hypersensitivity is due to the presence of amino acid duplication synthesis side product(s) which gives rise to novel T cell epitopes. HLA typing in allergic patients shows an enrichment of five particular HLA DRB1 alleles. Two of these alleles (DRB1*0701 and DRB1*1104) were shown to be able to bind the impurity rather than the drug. Using the WhIM algorithm, we have evaluated all possible amino acid duplication impurities for the presence of new T cell epitopes at both a population level and an individualized level. Out of these possible impurities, five create putative T cell neo-epitopes for the HLA- DR7 and/or DR11 families, and one or more of these could be contributing to the observed hypersensitivity to Taspoglutide in subjects with DRB1*0701 and/or DRB1*1104.
Poster ID# 4983
Poster ID# 5011
Effect of Octreotide-Bile Salt Interactions on Octreotide Flux Through Polymeric Membranes
ABSTRACT:It is well-known that components of the aqueous gastrointestinal environment e.g. bile acid mixed micelles (BAMM) affect the oral absorption of many small molecule drugs. Biorelevant media such as fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF) are now frequently used to characterize drug More dissolution and solubilization in vitro, especially for poorly water-soluble drugs. Despite the significant progress made for small molecule drugs, there is a lack of understanding pertaining to the impact of the aqueous gastrointestinal environment on peptide drug behavior (beyond enzymatic degradation). Many efforts towards achieving the oral delivery of peptide drugs have been made in recent years, and in 2018 oral semaglutide and octreotide drug products progressed to Phase III clinical trials. These scientific advances support that oral peptide pharmacotherapy is possible, however, an improved understanding of peptide-bile interactions in solution is essential especially given large inter- and intra-individual variability in bile acid concentrations in vivo which may be (in part) responsible for low and variable oral peptide bioavailability. Therefore, the major aim of this study was to investigate the interaction of peptide drug molecules with bile salts present in the gastrointestinal environment. Octreotide, a cyclic octapeptide, was used as a model drug. The interaction of octreotide with BAMM was investigated by studying octreotide flux through regenerated cellulose dialysis membranes in a side-by-side diffusion cell from biorelevant media e.g. FaSSIF/FeSSIF. Buffer without bile salts was used as a control, and octreotide concentrations in the receiver chamber were quantified using high performance liquid chromatography. Biorelevant media was found to have a significant impact on octreotide flux in vitro; at a donor concentration of 0.1 mM, octreotide mass transfer decreased from 34.2 ± 0.9 ng/min in pH 6.5 buffer to 27.6 ± 0.3 and 15.0 ± 0.1 ng/min in FaSSIF and FeSSIF, respectively. The impact of specific bile salts found in the human gastrointestinal environment (e.g. taurocholate, glycocholate, taurodeoxycholate) on octreotide flux was also systematically investigated at concentrations above and below the bile salt critical micelle concentration(s).
Poster ID# 4824
Flash Purification Methodology for Synthetic Peptides
ABSTRACT:Synthetic Peptides are common place in the world and are utilized for a wide range of functions including vaccines, control of diabetes and as affinity ligands in protein purification. Most common peptides are comprised of no more than 50 amino acids. Due to this reasonable number, they can be synthesized with ease and at a low cost. The More commonality and low cost of production drives the need for a quick, simple and cost effective purification method.
Flash chromatography is often used for quick purifications during intermediate steps of pharmaceutical product synthesis. These intermediate purifications allow for higher yields of subsequent steps and ultimately more product. Unlike LC columns, Flash cartridges are considered disposable. They require less cleaning and maintenance since they are often disposed of after 1-3 purification runs. Additionally, Flash systems typically have only basic functions, including low pressure pumps, a fraction collector and a UV-Vis detector. This simple setup, allows them to be easily maintained and purchased at a lower cost. Flash cartridges are available in many common stationary phase chemistries suitable for Reverse Phase and Normal Phases separations.
Presented here is a rapid 2-step process for purifying crude synthetic peptide material. First, the material is evaluated with a generic HPLC gradient method. Second, the result from the generic gradient method is used to create a Flash Chromatography method that can purify the material. This 2-step process is demonstrated with a 22aa crude synthetic peptide sample. The initial crude had a purity of 77.3% and depending on how the fractions were pooled, the purity could have been upgraded to 94.0% with a yield of 45.1% or 90.4% with a yield of 84.6%. This 2-step process took approximately 45 minutes to complete