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Matthew Welsch
Director of Chemical Biology, Kleo Pharmaceuticals Inc.

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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 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.

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 degrading signal for destroying the androgen receptor (AR) protein, the most critical driver in newly developed and post-Abiraterone/ Enzalutamide castration-resistant prostate 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 mammalian cell application. 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. Intratumoral 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 disrupting AR protein stability is feasible to be developed as a novel therapy for advanced prostate cancers.

Suresh Pitram
Senior Advisor, External Innovation, Elanco

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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 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.

Benyi Li MD PhD

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 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.

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 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.[1]
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.[2] 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

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. 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.