Purification of N-acetylgalactosamine-modified-oligonucleotides using orthogonal anion-exchange and mixed-mode chromatography approaches.

N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.

Identification and Optimization of a Minor Allele-Specific siRNA to Prevent PNPLA3 I148M-Driven Nonalcoholic Fatty Liver Disease.

Human genome wide association studies confirm the association of the rs738409 single nucleotide polymorphism (SNP) in the gene encoding protein patatin like phospholipase domain containing 3 () with nonalcoholic fatty liver disease (NAFLD); the presence of the resulting mutant PNPLA3 I148M protein is a driver of nonalcoholic steatohepatitis (NASH). While -deficient mice do not display an adverse phenotype, the safety of knocking down endogenous wild type in humans remains unknown. To expand the scope of a potential targeted NAFLD therapeutic to both homozygous and heterozygous rs738409 populations, we sought to identify a minor allele-specific small interfering RNA (siRNA).

Purification of guanine-quadruplex using monolithic stationary phase under ion-exchange conditions.

The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media-quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle-based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex.

Use of Cryopreserved Hepatocytes as Part of an Integrated Strategy to Characterize In Vivo Clearance for Peptide-Antibody Conjugate Inhibitors of Nav1.7 in Preclinical Species.

The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described.

Plasma and Liver Protein Binding of -Acetylgalactosamine-Conjugated Small Interfering RNA.

Understanding small interfering RNA (siRNA) fraction unbound ( ) in relevant physiologic compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of -acetylgalactosamine-conjugated siRNA using classic small-molecule in vitro techniques, we found that the hydrodynamic radius was critical in determining the size exclusion limit requirements for isolation, largely validating the siRNA "rigid rod" hypothesis. With this knowledge, we developed an orthogonally validated 50 kDa molecular-mass cutoff ultrafiltration assay to quantify in biologic matrices including human, nonhuman primate, rat, and mouse plasma, and human liver homogenate.

Engineering Na1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation To Enhance Pharmacokinetics.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel Na1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed Na1.

Discovery of Tarantula Venom-Derived Na1.7-Inhibitory JzTx-V Peptide 5-Br-Trp24 Analogue AM-6120 with Systemic Block of Histamine-Induced Pruritis.

Inhibitors of the voltage-gated sodium channel Na1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified Na1.

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.

The optimization of aminooxadiazoles as orally active inhibitors of Cdc7.

A series of aminooxadiazoles was optimized for inhibition of Cdc7. Early lead isoquinoline 1 suffered from modest cell potency (cellular IC50=0.71 μM measuring pMCM2), low selectivity against structurally related kinases, and high IV clearance in rats (CL=18 L/h/kg).

Optimization of a Potent, Orally Active S1P1 Agonist Containing a Quinolinone Core.

The optimization of a series of S1P1 agonists with limited activity against S1P3 is reported. A polar headgroup was used to improve the physicochemical and pharmacokinetic parameters of lead quinolinone 6. When dosed orally at 1 and 3 mg/kg, the azahydroxymethyl analogue 22 achieved statistically significant lowering of circulating blood lymphocytes 24 h postdose.

Quinolinone-based agonists of S1P₁: use of a N-scan SAR strategy to optimize in vitro and in vivo activity.

We reveal how a N-scan SAR strategy (systematic substitution of each CH group with a N atom) was employed for quinolinone-based S1P(1) agonist 5 to modulate physicochemical properties and optimize in vitro and in vivo activity. The diaza-analog 17 displays improved potency (hS1P(1) RI; 17: EC(50)=0.020 μM, 120% efficacy; 5: EC(50)=0.

4-Methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide: A Potent and Selective Agonist of S1P1.

The sphingosine-1-phosphate-1 receptor (S1P1) and its endogenous ligand sphingosine-1-phosphate (S1P) cooperatively regulate lymphocyte trafficking from the lymphatic system. Herein, we disclose 4-methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide (8), an uncommon example of a synthetic S1P1 agonist lacking a polar headgroup, which is shown to effect dramatic reduction of circulating lymphocytes (POC = -78%) in rat 24 h after a single oral dose (1 mg/kg). The excellent potency that 8 exhibits toward S1P1 (EC50 = 0.

Discovery of highly selective and potent p38 inhibitors based on a phthalazine scaffold.

Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38alpha including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gly110.

3-amino-7-phthalazinylbenzoisoxazoles as a novel class of potent, selective, and orally available inhibitors of p38alpha mitogen-activated protein kinase.

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors.

First X-ray cocrystal structure of a bacterial FabH condensing enzyme and a small molecule inhibitor achieved using rational design and homology modeling.

The first cocrystal structure of a bacterial FabH condensing enzyme and a small molecule inhibitor is reported. The inhibitor was obtained by rational modification of a high throughput screening lead with the aid of a S. pneumoniae FabH homology model.