Synthesis, Structure, and SAR of Tetrahydropyran-Based LpxC Inhibitors

In the search for novel Gram-negative agents, we performed a comprehensive search of the AstraZeneca collection and identified a tetrahydropyran-based matrix metalloprotease (MMP) inhibitor that demonstrated nanomolar inhibition of UDP-3-<i>O</i>-(acyl)-<i>N</i>-acetylglucosamine deacetylase (LpxC). Crystallographic studies in <i>Aquifex aeolicus</i> LpxC indicated the tetrahydropyran engaged in the same hydrogen bonds and van der Waals interactions as other known inhibitors. Systematic optimization of three locales on the scaffold provided compounds with improved Gram-negative activity. However, the optimization of LpxC activity was not accompanied by reduced inhibition of MMPs. Comparison of the crystal structure of the native product, UDP-3-<i>O</i>-(acyl)-glucosamine, in <i>Aquifex aeolicus</i> to the structure of a tetrahydropyran-based inhibitor indicates pathways for future optimization

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA

Intracellular delivery of mRNA holds great potential for vaccine− and therapeutic discovery and development. Despite increasing recognition of the utility of lipid-based nanoparticles (LNPs) for intracellular delivery of mRNA, particle engineering is hindered by insufficient understanding of endosomal escape, which is believed to be a main limiter of cytosolic availability and activity of the nucleic acid inside the cell. Using a series of CRISPR-based genetic perturbations of the lysosomal pathway, we have identified that late endosome/lysosome (LE/Ly) formation is essential for functional delivery of exogenously presented mRNA. Lysosomes provide a spatiotemporal hub to orchestrate mTOR signaling and are known to control cell proliferation, nutrient sensing, ribosomal biogenesis, and mRNA translation. Through modulation of the mTOR pathway we were able to enhance or inhibit LNP-mediated mRNA delivery. To further boost intracellular delivery of mRNA, we screened 212 bioactive lipid-like molecules that are either enriched in vesicular compartments or modulate cell signaling. Surprisingly, we have discovered that leukotriene-antagonists, clinically approved for treatment of asthma and other lung diseases, enhance intracellular mRNA delivery in vitro (over 3-fold, <i>p</i> < 0.005) and in vivo (over 2-fold, <i>p</i> < 0.005). Understanding LNP-mediated intracellular delivery will inspire the next generation of RNA therapeutics that have high potency and limited toxicity

Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines

mRNA vaccines have the potential to tackle many unmet medical needs that are unable to be addressed with conventional vaccine technologies. A potent and well-tolerated delivery technology is integral to fully realizing the potential of mRNA vaccines. Pre-clinical and clinical studies have demonstrated that mRNA delivered intramuscularly (IM) with first-generation lipid nanoparticles (LNPs) generates robust immune responses. Despite progress made over the past several years, there remains significant opportunity for improvement, as the most advanced LNPs were designed for intravenous (IV) delivery of siRNA to the liver. Here, we screened a panel of proprietary biodegradable ionizable lipids for both expression and immunogenicity in a rodent model when administered IM. A subset of compounds was selected and further evaluated for tolerability, immunogenicity, and expression in rodents and non-human primates (NHPs). A lead formulation was identified that yielded a robust immune response with improved tolerability. More importantly for vaccines, increased innate immune stimulation driven by LNPs does not equate to increased immunogenicity, illustrating that mRNA vaccine tolerability can be improved without affecting potency. Keywords: mRNA, vaccines, LNP, intramuscular, lipids, tolerability, immunogenicity, formulatio

Dual mRNA therapy restores metabolic function in long-term studies in mice with propionic acidemia

Propionic acidemia is a serious pediatric inherited disorder with no effective treatments. Here the authors demonstrate that delivering dual mRNAs as an enzyme replacement approach can be used as an effective therapy in a mouse model of propionic acidemia, with potential applicability to chronically administer multiple mRNAs in other genetic disorders

Discovery of Efficacious Pseudomonas aeruginosa-Targeted Siderophore-Conjugated Monocarbams by Application of a Semi-Mechanistic PK/PD Model

In order to identify new agents for the treatment of Pseudomonas aeruginosa infections to address the serious threat to society posed by the evolution of multi-drug resistant P. aeruginosa, we focused on the well established family of Beta-lactams antibiotics. There is evidence they are effective against the target pathogen and their resistance profiles and pharmacology are well established. To address the major resistance mechanisms to other Beta-lactam antibiotics we studied siderophore-conjugated monocarbams. This class of monocyclic Beta-lactams is stable to metallo Beta-lactamases and they have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of the Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. We examined modifications to the monocarbam linker, the siderophore, and the oxime portion of the molecules. Through these efforts we identified a series of pyrrolidinone-based monocarbams which have good P. aeruginosa cellular activity (P. aeruginosa MIC90 = 2 g/ml), excellent free fraction levels (> 20 % free) and good hydrolytic stability (t1/2 ≥ 100 h). In order to differentiate our compounds and enable prioritization for future in vivo studies, we developed a robust mechanistic PK/PD model which enables prediction of in vivo efficacy from in vitro data

SAR and Structural Analysis of Siderophore-Conjugated Monocarbam Inhibitors of <i>Pseudomonas aeruginosa</i> PBP3

A main challenge in the development of new agents for the treatment of <i>Pseudomonas aeruginosa</i> infections is the identification of chemotypes that efficiently penetrate the cell envelope and are not susceptible to established resistance mechanisms. Siderophore-conjugated monocarbams are attractive because of their ability to hijack the bacteria’s iron uptake machinery for transport into the periplasm and their inherent stability to metallo-β-lactamases. Through development of the SAR we identified a number of modifications to the scaffold that afforded active anti-<i>P. aeruginosa</i> agents with good physicochemical properties. Through crystallographic efforts we gained a better understanding into how these compounds bind to the target penicillin binding protein PBP3 and factors to consider for future design

Discovery of Efficacious Pseudomonas aeruginosa-Targeted Siderophore-Conjugated Monocarbams by Application of a Semi-mechanistic Pharmacokinetic/Pharmacodynamic Model

To identify new agents for the treatment of multi-drug-resistant Pseudomonas aeruginosa, we focused on siderophore-conjugated monocarbams. This class of monocyclic β-lactams are stable to metallo-β-lactamases and have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker, siderophore, and oxime portion of the molecules were examined. Through these efforts, a series of pyrrolidinone-based monocarbams with good P. aeruginosa cellular activity (P. aeruginosa MIC₉₀ = 2 μg/mL), free fraction levels (>20% free), and hydrolytic stability (<i>t</i>_1/2 ≥ 100 h) were identified. To differentiate the lead compounds and enable prioritization for in vivo studies, we applied a semi-mechanistic pharmacokinetic/pharmacodynamic model to enable prediction of in vivo efficacy from in vitro data

Systemic messenger RNA as an etiological treatment for acute intermittent porphyria

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP