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A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 months. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease., Competing Interests: Competing interestsAt the time the work described in this manuscript was performed, H.D.K., R.W., K.S., P.D., R.D., C.J.W., and G.J.N. all worked for Sanofi., (© The Author(s) 2020.)

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intranasal drug delivery is a noninvasive drug delivery route that can enhance systemic delivery of therapeutics with poor oral bioavailability by exploiting the rich microvasculature within the nasal cavity. The intranasal delivery route has also been targeted as a method for improved brain uptake of neurotherapeutics, with a goal of harnessing putative, direct nose-to-brain pathways. Studies in rodents, nonhuman primates, and humans have pointed to the efficacy of intranasally delivered neurotherapeutics, while radiolabeling studies have analyzed brain uptake following intranasal administration. In the present study, we employed carbon-11 radioactive methylation to assess the pharmacokinetic mechanism of intranasal delivery of Orexin A, a native neuropeptide and prospective antinarcoleptic drug that binds the orexin receptor 1. Using physicochemical and pharmacological analysis, we identified the methylation sites and confirmed the structure and function of methylated Orexin A (CH 3 -Orexin A) prior to monitoring its brain uptake following intranasal administration in rodent and nonhuman primate. Through positron emission tomography (PET) imaging of [ 11 C]CH 3 -Orexin A, we determined that the brain exposure to Orexin A is poor after intranasal administration. Additional ex vivo analysis of brain uptake using [ 125 I]Orexin A indicated intranasal administration of Orexin A affords similar brain uptake when compared to intravenous administration across most brain regions, with possible increased brain uptake localized to the olfactory bulbs.

There has been a resurgence of interest in peptide pharmaceuticals as they have an advantage of potency, selectivity and less toxicity compared with small-molecule therapeutics. The main draw back of peptides is lack of stability to biological media. Constraining a peptide has been one of the approaches to improving in vivo stability of the peptides. Several new modalities in constraining peptides have been developed over recent years and this review highlights some of the new developments. The newer cyclization strategies have rendered, in some cases, oral activity, cell permeability, improved potency at the target receptor, selectivity against receptor subtypes and improved stability to enzymes. As chemists further understand the rules governing cell permeability, oral absorption and enhancing stability of peptides, we can expect to see more peptides entering clinic for many unmet medical needs.

Beginning with a screening hit, unique thienopyrazole-indole inhibitors of Itk (interleukin-2-inducible tyrosine kinase) were designed, synthesized, and crystallized in the target kinase. Although initial compounds were highly active in Itk, they were not selective. Increasing the steric bulk around a tertiary alcohol at the 5-indole position dramatically improved selectivity toward Lyk and Syk, but not Txk. Substitutions at the 3- and 4-indole positions gave less active compounds that remained poorly selective. A difluoromethyl substitution at the 5-position of the thienopyrazole led to a highly potent and selective compound. Phenyl at this position reduced activity and selectivity while pushing the side-chains of Lys-391 and Asp-500 away from the binding pocket. Novel and selective thienopyrazole inhibitors of Itk were designed as a result of combining structure-based design and medicinal chemistry., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The synthesis and biological activities of seven new glucagon analogues are reported. The design of compounds 2-5 is based on potent antagonists recently reported from this laboratory, where we have focused on modifications in the N-terminal region. In this report we have concentrated specifically on modifications to histidine-1. In addition we have prepared two cyclic compounds 7 and 8, related to a linear in vivo antagonist [Glu9]glucagon, reported by Merrifield (Unson et al. (1987) Proc. Natl. Acad. Sci. USA 84, 4083-4087). The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue (S)-5,6,7,8-tetrahydro-5-oxoimidazo(1,5-c)pyrimidine-7-carboxylic acid (Toc), desaminohistidine (dHis) and 3-(4-nitrobenzyl)histidine. The structures of the new compounds are as follows. [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (2); [Toc1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon amide (3); [3-(4-nitrobenzyl)His1,D-Phe4,Tyr5,Arg12,Lys17,18,G lu21]glucagon (4); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (5); [dHis1,Glu9]glucagon (6); (desHis1)[Glu9,Lys12]glucagon amide (7); (desHis1)-[Glu9,Lys12,Asp15]glucagon amide (8). The binding potencies of the linear analogues, as expressed a percentage of glucagon binding, are 2.6 (2), 0.13 (3), 0.8 (4), 0.8 (5), 2.2 (6). Both cyclic analogues 7 and 8 show biphasic binding curves. The IC50 values for 7 at the high and low affinity sites are 1.5 and 167 nM, respectively (IC50 of glucagon = 1.3 nM). The IC50 values for 8 at the high and low affinity sites are 4.7 and 3451 nM, respectively. The cyclic analogues are characterized by fast atom bombardment mass spectrometry of endoproteinase ASP-N digests. The specificity of the enzyme used in these studies enables differentiation of isomers of the cyclic glucagon analogues which differ only in the position of cyclic amide bond. Analogues 2, 3 and 5-8 are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase (AC) system. Analogue 4 is a partial agonist (5.7% compared to glucagon) of AC. Introduction of unusual amino acids which do not contain a primary alpha-amino group such as Toc at the N-terminus is expected to increase in vivo metabolic stability by protecting against degradation by aminopeptidases.

Fast atom bombardment mass spectral mapping of endoproteinase Asp-N digest mixtures is used for characterization of new synthetic linear and cyclic glucagon analogs. The results allow rapid identification of sequence modifications in linear glucagon analogs. For the cyclic compounds, the technique allows confirmation of the presence and position of the cyclic amide bond, as well as verification of the sequence of the modified glucagon analogs. The specificity of the Asp-N enables differentiation of isometric glucagon analogs which differ only in the position of the cyclic amide bond. Important information concerning the purity of the synthetic analogs is also available.