Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA.

Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA).

When Global Becomes Virtual: A Survey of Virtual Global Health Education Activities During the COVID-19 Pandemic Among Pediatric Educators.

Background: During the COVID-19 pandemic, global health education activities were disrupted. Transitioning to virtual options has allowed educators and trainees to continue global health education and partnerships, though the acceptability and implementation of this transition is unknown.

Objective: To evaluate current and planned virtual global health education activities (VGHEAs) of a group of US global health educators during the COVID-19 pandemic and to assess perceived benefits and challenges of VGHEAs.

Design and biological characterization of a series of dual mechanism ERK1/2 inhibitors with a Triazolopyridinone core.

Oncology clinical development programs have targeted the RAS/RAF/MEK/ERK signaling pathway with small molecule inhibitors for a variety of cancers during the past decades, and most therapies have shown limited or minimal success. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, most cancers have shown treatment resistance after several months of inhibitor usage, and reports indicate resistance is often associated with the reactivation of the MAPK signaling pathway.

Detection of SARS-CoV-2 in exhaled air using non-invasive embedded strips in masks.

Background: SARS-CoV-2 emerged in 2019 and resulted in a pandemic causing millions of infections worldwide. Gold-standard for SARS-CoV-2 detection uses quantitative RT-qPCR on respiratory secretions to detect viral RNA (vRNA). Acquiring these samples is invasive, can be painful for those with xerostomia and other health conditions, and sample quality can vary greatly.

Harmonization of Multiple SARS-CoV-2 Reference Materials Using the WHO IS (NIBSC 20/136): Results and Implications.

Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms.

Perceived roles, benefits and barriers of virtual global health partnership initiatives: a cross-sectional exploratory study.

Background: Virtual global health partnership initiatives (VGHPIs) evolved rapidly during the COVID-19 pandemic to ensure partnership continuity. However the current landscape for VGHPI use and preference is unknown. This study aimed to increase understanding of GH partners' perspectives on VGHPIs.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020.

Antimicrobial Resistance Trends at a Pediatric Hospital in Guatemala City, 2005-2019.

Antimicrobial resistance (AMR) is increasing worldwide. We analyzed AMR rates for bacterial species identified from pediatric blood cultures between 2005 and 2019 at a single institution in Guatemala. We found significantly increased rates in Gram-negative resistance, with a high prevalence of carbapenem-resistant Acinetobacter and Klebsiella harboring the New Delhi metallo-beta-lactamase gene.

Regulation of Lipoprotein Homeostasis by Self-Assembling Peptides.

High levels of serum low-density lipoprotein (LDL) cholesterol contribute to atherosclerosis, a key risk factor of cardiovascular diseases. PCSK9 is a circulatory enzyme that downregulates expression of hepatic LDL receptors, concomitantly increasing serum LDL-C. This work investigates a small, self-assembling peptide, EPep2-8, as a peptide inhibitor of PCSK9.

MK-8353: Discovery of an Orally Bioavailable Dual Mechanism ERK Inhibitor for Oncology.

The emergence and evolution of new immunological cancer therapies has sparked a rapidly growing interest in discovering novel pathways to treat cancer. Toward this aim, a novel series of pyrrolidine derivatives (compound ) were identified as potent inhibitors of ERK1/2 with excellent kinase selectivity and dual mechanism of action but suffered from poor pharmacokinetics (PK). The challenge of PK was overcome by the discovery of a novel 3()-thiomethyl pyrrolidine analog .

Discovery of 3(S)-thiomethyl pyrrolidine ERK inhibitors for oncology.

Compound 5 (SCH772984) was identified as a potent inhibitor of ERK1/2 with excellent selectivity against a panel of kinases (0/231 kinases tested @ 100 nM) and good cell proliferation activity, but suffered from poor PK (rat AUC PK @10 mpk = 0 μM h; F% = 0) which precluded further development. In an effort to identify novel ERK inhibitors with improved PK properties with respect to 5, a systematic exploration of sterics and composition at the 3-position of the pyrrolidine led to the discovery of a novel 3(S)-thiomethyl pyrrolidine analog 28 with vastly improved PK (rat AUC PK @10 mpk = 26 μM h; F% = 70).

Discovery of hydroxyaniline amides as selective Extracellular Regulated Kinase (Erk) inhibitors.

Starting from weak μM hits identified through affinity based Automated Ligand Identification System (ALIS) screenings, double digit nM hydroxyaniline amide Erk inhibitors were discovered. This class of compounds had the unique dual mechanism of inhibiting activated and non-activated forms of Erk. They generally had high degree of selectivity in kinase panel tested.

Discovery of novel, dual mechanism ERK inhibitors by affinity selection screening of an inactive kinase.

An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop.

Modulating the interaction between CDK2 and cyclin A with a quinoline-based inhibitor.

A new class of quinoline-based kinase inhibitors has been discovered that both disrupt cyclin dependent 2 (CDK2) interaction with its cyclin A subunit and act as ATP competitive inhibitors. The key strategy for discovering this class of protein-protein disrupter compounds was to screen the monomer CDK2 in an affinity-selection/mass spectrometry-based technique and to perform secondary assays that identified compounds that bound only to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (Kd) of the original hit improved from 1 to 0.

In vitro kinetic profiling of hepatitis C virus NS3 protease inhibitors by progress curve analysis.

Kinetic profiling of drug binding to its target reveals important mechanistic parameters including drug-target residence time. In this chapter, we focus on global progress curve analysis as a convenient method for kinetic profiling. Detailed guidelines with pros and cons for various experimental designs and data analysis are provided.

Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors.

The high frequency of activating RAS or BRAF mutations in cancer provides strong rationale for targeting the mitogen-activated protein kinase (MAPK) pathway. Selective BRAF and MAP-ERK kinase (MEK) inhibitors have shown clinical efficacy in patients with melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the extracellular signal-regulated kinase (ERK) signaling pathway.

Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor.

Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC(50) values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters.

3-phosphoinositide-dependent protein kinase-1 regulates proliferation and survival of cancer cells with an activated mitogen-activated protein kinase pathway.

Engagement of cell surface receptor tyrosine kinases by insulin and growth factors activates phosphatidylinositol 3-kinase (PI3K) and generates the second messenger, phosphatidylinositol 3,4,5-trisphosphate. This second messenger leads to the recruitment of 3-phosphoinositide-dependent protein kinase-1 (PDK1) to the proximal side of the plasma membrane, which results in the activation of AKT kinase. In addition, PDK1 can phosphorylate numerous other kinases, including p90RSK, a kinase downstream of mitogen-activated protein kinase (MAPK) that is important for cell proliferation and survival.

Structure-guided discovery of cyclin-dependent kinase inhibitors.

CDK2 inhibitors containing the related bicyclic heterocycles pyrazolopyrimidines and imidazopyrazines were discovered through high-throughput screening. Crystal structures of inhibitors with these bicyclic cores and two more related ones show that all but one have a common binding mode featuring two hydrogen bonds (H-bonds) to the backbone of the kinase hinge region. Even though ab initio computations indicated that the imidazopyrazine core would bind more tightly to the hinge, pyrazolopyrimidines gain an advantage in potency through participation of N4 in an H-bond network involving two catalytic residues and bridging water molecules.

Method for quantitative protein-ligand affinity measurements in compound mixtures.

This manuscript describes an affinity selection-mass spectrometry (AS-MS) method for quantitative protein-ligand binding affinity (Kd) measurements in large compound libraries. The ability of a titrant ligand to displace a target-bound library member-as measured by MS-reveals the affinity ranking of the mixture component relative to "internal affinity calibrants", compounds of known affinity for the target. This technique does not require that the precise concentration of each ligand is known; therefore, unpurified products of mixture-based combinatorial synthesis may be used for affinity optimization and developing structure-activity relationships.

Thermodynamics of nucleotide and non-ATP-competitive inhibitor binding to MEK1 by circular dichroism and isothermal titration calorimetry.

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. A wealth of information about the enzymatic activity of MEK1, its domain functions, and inhibitor action is available; however, the thermodynamic properties of the interaction between MEK1 and ligands, such as nucleotides and non-ATP-competitive inhibitors, have not been reported. This study describes the thermodynamic parameters for the binding interactions of MEK1, nucleotides, and non-ATP-competitive inhibitor complexes using temperature-dependent circular dichroism (TdCD) and isothermal titration calorimetry (ITC).

Expression and purification of phosphorylated and non-phosphorylated human MEK1.

Kinases exist in either a high or low activity form depending on the phosphorylation state of the activating lip. These two different forms of the same kinase may adopt different conformations that affect not only activity but also inhibitor binding and the ability to crystallize the protein. Therefore, isolation of homogenous preparations of the phosphorylated and non-phosphorylated versions of a kinase is critical for accurate biophysical measurements of activity, stability and ligand binding as well as for protein crystallization.

Ligand binding affinity determined by temperature-dependent circular dichroism: cyclin-dependent kinase 2 inhibitors.

To support drug discovery efforts for cyclin-dependent kinase 2 (CDK2), a moderate-throughput binding assay that can rank order or estimate the affinity of lead inhibitors has been developed. The method referred to as temperature-dependent circular dichroism (TdCD) uses the classical temperature-dependent unfolding of proteins by circular dichroism (CD) to measure the degree of protein unfolding in the absence and presence of potential inhibitors. The midpoint of unfolding is the Tm value.

Protein farnesyltransferase inhibitors exhibit potent antimalarial activity.

New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal drug target.