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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., Graphical Abstract

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer's disease.

Attempts to optimize pharmacokinetic properties in a promising series of pyrrolopyrimidinone MARK inhibitors for the treatment of Alzheimer's disease are described. A focus on physical properties and ligand efficiency while prosecuting this series afforded key tool compounds that revealed a large discrepancy in the rat in vitro-in vivo DMPK (Drug Metabolism/Pharmacokinetics) correlation. These differences prompted an in vivo rat disposition study employing a radiolabeled representative of the series, and the results from this experiment justified the termination of any further optimization efforts.

Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.

The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.

The concept of mRNA as a therapeutic platform has historically been ignored owing to challenges in oligonucleotide delivery and, maybe more importantly, the perceived shortcomings of mRNA with regard to stability and immunogenicity. Advances in several areas have recently prompted a reexamination of such dogma. Significant improvements in oligonucleotide delivery have been realized over the past decade and their application to mRNA has enabled a more rapid path toward clinical development of this new modality. Similarly, recent discoveries in mRNA chemistry further enhance the attractiveness of this platform by attenuating innate immune activation and maximizing protein expression. With these advances, mRNA is positioned to become an important new therapeutic modality.

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.

We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.

Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAC1 and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1-selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

An efficient procedure for the preparation of trans-2,6-diaryl piperidinones has been developed. Addition of aryl Grignard reagents to 2-aryl dihydropyridones under catalytic copper promoted conditions generates the trans isomer exclusively, an unprecedented stereochemical event. The X-ray structures of both starting material and product have been solved and shed light on the steric constraints and substrate geometry leading to the observed product. The reaction conditions tolerate a variety of aromatic nucleophiles to generate C2-symmetric products in good overall yields.

A series of low-molecular weight 2,6-diamino-isonicotinamide BACE-1 inhibitors containing an amine transition-state isostere were synthesized and shown to be highly potent in both enzymatic and cell-based assays. These inhibitors contain a trans-S,S-methyl cyclopropane P3 which bind BACE-1 in a 10s-loop down conformation giving rise to highly potent compounds with favorable molecular weight and moderate to high susceptibility to P-glycoprotein (P-gp) efflux.

Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer’s disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

beta-Secretase inhibition offers an exciting opportunity for therapeutic intervention in the progression of Alzheimer's disease. A series of isonicotinamides derived from traditional aspartyl protease transition state isostere inhibitors has been optimized to yield low nanomolar inhibitors with sufficient penetration across the blood-brain barrier to demonstrate beta-amyloid lowering in a murine model.

The catalytic effect of alkali-metal tert-butoxide clusters on the rate of ester interchange for several pairs of esters has been determined in nonpolar and weakly polar solvents. Reactivities increase in the order (Li+ < Na+ < K+ < Rb+ < Cs+) with the fastest rates reaching 107 catalytic turnovers per hour (TO/h). Ester interchange rates were sensitive to the size of both the transferring OR groups and the ester substituent. Phenyl esters did not exchange with aliphatic esters due to nonstatistical breakdown patterns in the tetrahedral intermediate. A first-order equilibration analysis on the interchange between tert-butyl acetate (tBuAc) and methyl benzoate (MeBz) (5 mol % NaOtBu) indicated enhanced reaction rates as the reaction proceeded. Isolation and quenching (DCl/D2O) of precipitated catalyst points to a mechanism whereby sequential methoxy incorporation into the catalyst cluster increases activity, but eventually precipitates out of solution as a 3:1 OMe:OtBu cluster. The rate law was determined ...

The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.

In order to rapidly screen and select lead candidates for in vivo evaluation of lipid nanoparticles (LNPs) for systemic small interfering RNA (siRNA) delivery, an in vitro assay amenable to high-throughput screening (HTS) is developed. The strategy is to mimic the in vivo experience of LNPs after systemic administration, such as interactions with serum components, exposure to endosomal pH environments, and interactions with endosomal membrane lipids. It is postulated that the amount of siRNA released from LNPs after going through these treatments can be used as a screening tool to rank order the effectiveness of siRNA delivery by lipid nanoparticles in vivo. LNPs were incubated with 50% serum from different species (i.e. mouse, rat, or rhesus) at 37°C. The resulting samples were then reacted with anionic, endosomal-mimicking lipids at different pHs. The amount of siRNA released from LNPs was determined using spectrophotometry employing the fluorescent indicator SYBR Gold. Our results indicated that the amount of siRNA liberated was highly dependent upon the species of serum used and the pH to which it was exposed. LNPs treated with mouse serum showed higher levels of siRNA release, as did those subjected to endosomal pH (6.0), compared to physiological pH. Most interestingly, a good correlation between the amount of siRNA released and the in vivo efficacy was observed. In conclusion, an in vitro siRNA release assay was developed to screen and rank order LNPs for in vivo evaluation.

Transgenic mice with Tie2- green fluorescent protein (GFP) are used as a model to study the kinetic distribution of the Cy5-siRNA delivered by lipid nanoparticles (LNP) into the liver. After the mouse is injected with the LNP, it undergoes a procedure of intra-vital multi-photon microscopy imaging over a period of two hours, during which the process for the nanoparticle to diffuse into the hepatocytes from the vasculature system is monitored. Since the images are obtained in-vivo, the quantification of Cy5 kinetics suffers from the moving field of view (FOV). A method is proposed to register the sequence of images through template matching. Based on the semi-automatic segmentations of the vessels in the common FOV, the registered images are segmented into three regions of interest (ROI) in which the Cy5 signals are quantified. Computation of the percentage signal strength in the ROIs over time allows for the analysis of the diffusion of Cy5-siRNA into the hepatocytes, and helps demonstrate the effectiveness of the Cy5-siRNA delivery vehicle.

We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.

This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.

Starting from a 2-amino-6-methylpyridine P1 group and following a strategy of enlarging it whilst reducing its polarity, we have developed a series of potent, moderately basic azaindoles which are intrinsically much more selective for thrombin versus trypsin. Certain pyrazinone acetamide azaindole derivatives have pharmacokinetic parameters after oral administration to dogs, or efficacy in vitro, comparable to an optimized pyrazinone acetamide 2-amino-6-methylpyridine derivative.

A concise total synthesis of the potent antitumor macrolide (-)-laulimalide is described. The observation that homoallylic (or latent homoallylic) C-O bonds are present at C5, C9, C15, C19, and C23 led to the strategic decision to rely heavily on the asymmetric glycolate alkylation to construct both the C1-C14 fragment 3 and the C15-C27 subunit 4. A diastereoselective addition of a C1-C14 allylstannane to a C15-C27 alpha,beta-epoxyaldehyde served to join the two advanced fragments. A Mitsunobu macrolactonization of hydroxy acid 2 avoided isomerization of the sensitive 2,3-Z-enoate, which has been observed in base-catalyzed macrolactonizations. Removal of two TBS protecting groups to reveal the C15 and C20 hydroxyls occurred without rearrangement to isolaulimalide.

The histone deacetylase (HDAC) metalloenzymes are intricately involved in gene expression through epigenetic regulation of histone acetylation. They also regulate the acetylation status of numerous non-histone proteins such as transcription factors p53, STAT1 and NF-κB as well as α-tubulin, Hsp90 and Ku70. Of the eleven zinc-dependent HDAC enzymes identified, HDACs 1 and 2 appear to be most critical in oncogenesis and tumor maintenance. They are overexpressed in many human cancers and RNAi knockdown leads to increased apoptosis. We recently disclosed a family of novel HDAC1/HDAC2-selective biaryl inhibitors. In this presentation, we will describe unique features of these biaryl inhibitors that contribute to improved preclinical efficacy and tolerability. Desirable HDAC inhibitor properties identified from preclinical experience with Zolinza™ include subtype selectivity toward HDAC1/HDAC2 and prolonged target inhibition. Compelling in vitro and in vivo data indicates that solid tumor cell lines are most sensitive to HDAC inhibition under continuous exposure rather than intermittent exposure. These biaryl inhibitors exhibit extended target engagement in vivo, and are well tolerated in nude mice. Unlike other known HDAC inhibitors, these compounds exhibit a delay in and prolongation of histone hyperacetylation in nude mice bearing HCT116 tumors, extending beyond plasma clearance of the drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5433.