11 research outputs found
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Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.
CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo
Acyl Glucuronide Metabolites of 6‑Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]‑1<i>H</i>‑indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK)
Studies on indole-3-carboxylic acid
derivatives as direct activators
of human adenosine monophosphate-activated protein kinase (AMPK) α1β1γ1
isoform have culminated in the identification of PF-06409577 (<b>1</b>), PF-06885249 (<b>2</b>), and PF-06679142 (<b>3</b>) as potential clinical candidates. Compounds <b>1</b>–<b>3</b> are primarily cleared in animals and humans via glucuronidation.
Herein, we describe the biosynthetic preparation, purification, and
structural characterization of the glucuronide conjugates of <b>1</b>–<b>3</b>. Spectral characterization of the
purified glucuronides M1, M2, and M3 indicated that they were acyl
glucuronide derivatives. In vitro pharmacological evaluation revealed
that all three acyl glucuronides retained selective activation of
β1-containing AMPK isoforms. Inhibition of de novo lipogenesis
with representative parent carboxylic acids and their respective acyl
glucuronide conjugates in human hepatocytes demonstrated their propensity
to activate cellular AMPK. Cocrystallization of the AMPK α1β1γ1
isoform with <b>1</b>–<b>3</b> and M1–M3
provided molecular insights into the structural basis for AMPK activation
by the glucuronide conjugates
Spirocyclic Sulfamides as β‑Secretase 1 (BACE-1) Inhibitors for the Treatment of Alzheimer’s Disease: Utilization of Structure Based Drug Design, WaterMap, and CNS Penetration Studies To Identify Centrally Efficacious Inhibitors
β-Secretase 1 (BACE-1) is an attractive therapeutic
target for the treatment and prevention of Alzheimer’s disease
(AD). Herein, we describe the discovery of a novel class of BACE-1
inhibitors represented by sulfamide <b>14g</b>, using a medicinal
chemistry strategy to optimize central nervous system (CNS) penetration
by minimizing hydrogen bond donors (HBDs) and reducing P-glycoprotein
(P-gp) mediated efflux. We have also taken advantage of the combination
of structure based drug design (SBDD) to guide the optimization of
the sulfamide analogues and the in silico tool WaterMap to explain
the observed SAR. Compound <b>14g</b> is a potent inhibitor
of BACE-1 with excellent permeability and a moderate P-gp liability.
Administration of <b>14g</b> to mice produced a significant,
dose-dependent reduction in central Aβ<sub>X‑40</sub> levels
at a free drug exposure equivalent to the whole cell IC<sub>50</sub> (100 nM). Furthermore, studies of the P-gp knockout mouse provided
evidence that efflux transporters affected the amount of Aβ
lowering versus that observed in wild-type (WT) mouse at an equivalent
dose
Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro
Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (K-D = 0.6 mu M) compared with wild-type EGF-A (K-D = 1.2 mu M), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay
Discovery of Fragment-Derived Small Molecules for in Vivo Inhibition of Ketohexokinase (KHK)
Increased
fructose consumption and its subsequent metabolism have
been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin
resistance in humans. Since ketohexokinase (KHK) is the principal
enzyme responsible for fructose metabolism, identification of a selective
KHK inhibitor may help to further elucidate the effect of KHK inhibition
on these metabolic disorders. Until now, studies on KHK inhibition
with small molecules have been limited due to the lack of viable in
vivo pharmacological tools. Herein we report the discovery of <b>12</b>, a selective KHK inhibitor with potency and properties
suitable for evaluating KHK inhibition in rat models. Key structural
features interacting with KHK were discovered through fragment-based
screening and subsequent optimization using structure-based drug design,
and parallel medicinal chemistry led to the identification of pyridine <b>12</b>
Discovery and Preclinical Characterization of 6‑Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]‑1<i>H</i>‑indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy
Adenosine
monophosphate-activated protein kinase (AMPK) is a protein
kinase involved in maintaining energy homeostasis within cells. On
the basis of human genetic association data, AMPK activators were
pursued for the treatment of diabetic nephropathy. Identification
of an indazole amide high throughput screening (HTS) hit followed
by truncation to its minimal pharmacophore provided an indazole acid
lead compound. Optimization of the core and aryl appendage improved
oral absorption and culminated in the identification of indole acid,
PF-06409577 (<b>7</b>). Compound <b>7</b> was advanced
to first-in-human trials for the treatment of diabetic nephropathy
Discovery and Optimization of a Novel Spiropyrrolidine Inhibitor of β-Secretase (BACE1) through Fragment-Based Drug Design
Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5′-Adenosine Monophosphate-Activated Protein Kinase (AMPK)
Optimization
of the pharmacokinetic (PK) properties of a series
of activators of adenosine monophosphate-activated protein kinase
(AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic
acid clinical candidate (<b>1</b>) were examined with the goal
of reducing glucuronidation rate and minimizing renal excretion. Compounds <b>10</b> (PF-06679142) and <b>14</b> (PF-06685249) exhibited
robust activation of AMPK in rat kidneys as well as desirable oral
absorption, low plasma clearance, and negligible renal clearance in
preclinical species. A correlation of in vivo renal clearance in rats
with in vitro uptake by human and rat renal organic anion transporters
(human OAT/rat Oat) was identified. Variation of polar functional
groups was critical to mitigate active renal clearance mediated by
the Oat3 transporter. Modification of either the 6-chloroindole core
to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted
5-(3-pyridyl) group provided improved metabolic stability while minimizing
propensity for active transport by OAT3