Protein therapeutics can be re-engineer as brain penetrating IgG-fusion proteins for the CNS treatment of rare disorders, like Lysosomal Storage Disorders (LSD). Lysosomal enzymes, such as iduronase (IDUA) and sulfatases, are large molecule drugs that do not cross the blood-brain barrier (BBB). The BBB-penetration of enzyme therapeutics is enabled by re-engineering the recombinant enzyme as bi-functional IgG fusion proteins, wherein the IgG domain targets a specific endogenous receptor-mediated transporter system within the BBB, such as the human insulin receptor (HIR). The enzyme therapeutic domain of the fusion protein exerts the pharmacological effect in brain once across the BBB. Several brain penetrating IgG-fusion proteins have been engineered and validated. First in human proof of concept phase II clinical trial in LSD will be discussed.
Note that this is scheduled for oral presentation in the Scientific Session 2: Drug Delivery, Wednesday September 26, 2018, 9:30-12:30. Thanks
Discovery of novel peptide dendrimers with potent and broad spectrum of antimicrobial activity
Thissa N. Siriwardena and Jean-Louis Reymond*
University of Bern
Multidrug resistant (MDR) Pseudomonas aeruginosa and Acinetobacter baumannii have been listed, together with Enterobacteriaceae, as the most critical human pathogens by the World Health Organization in 2017. New antibiotics are urgently needed to address MDR bacteria. We recently reported antimicrobial peptide dendrimer (AMPD) G3KL, showing good activity against P. aeruginosa and other Gram-negative strains including MDR clinical isolates.1 We later optimized this dendrimer to a smaller lipidated analog, TNS18, which showed excellent activity against both gram negative and positive bacteria and was also active in a murine infection model against MDR clinical isolates of A. baumannii and E. coli.2 Here we report our latest progress in identifying new AMPD exploiting the concept of chemical space borrowed from small molecule cheminformatics and recently exemplified with bicyclic peptides.3 Our exploration of the dendrimer chemical space led us to the discovery of a more potent analogs of G3KL with an expanded activity range, improved serum stability and very good activity in an in vivo infection model.
1. Stach, M.; Siriwardena, T. N.; Kohler, T.; van Delden, C.; Darbre, T.; Reymond, J. L. Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug-Resistant Pseudomonas Aeruginosa. Angew. Chem., Int. Ed. 2014, 53, 12827-12831.
2. Siriwardena, T. N.; Stach, M.; He, R.; Gan, B. H.; Javor, S.; Heitz, M.; Ma, L.; Cai, X.; Chen, P.; Wei, D.; Li, H.; Ma, J.; Kohler, T.; van Delden, C.; Darbre, T.; Reymond, J. L. Lipidated Peptide Dendrimers Killing Multidrug-Resistant Bacteria. J. Am. Chem. Soc. 2018, 140, 423-432.
3. Di Bonaventura, I.; Jin, X.; Visini, R.; Probst, D.; Javor, S.; Gan, B.-H. ; Michaud, G.; Natalello, A.; Doglia, S. M.; Kohler, T.; van Delden, C. ; Stocker, A.; Darbre, T.; Reymond, J.-L.; Chem. Sci. 2017, 8, 6784-6798.
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.