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Structure-based design, synthesis and biological evaluation of a series of peptidomimetic β-secretase inhibitors incorporating hydroxyethylamine isosteres are described. We have identified inhibitor 24 which has shown exceedingly potent activity in memapsin 2 enzyme inhibitory (K i 1.8 nM) and cellular (IC 50 = 1 nM in Chinese hamster cells) assays. Inhibitor 24 has also shown very impressive in vivo properties (up to 65% reduction of plasma Aβ) in transgenic mice. The X-ray structure of protein-ligand complexes of memapsin 2 revealed critical interactions in the memapsin 2 active site. KeywordsAlzheimer; design; synthesis; inhibitor; memapsin 2; secretase Memapsin 2 (β-secretase, BACE 1) has become a major target for the development of inhibitor drugs for the treatment of Alzheimers's Disease (AD). 1 Memapsin 2 is the first protease that cleaves β-amyloid precursor protein (APP) in the pathway leading to the generation of amyloid-β peptide (Aβ) in the brain. 2,3 The excess level of Aβ results in the formation of amyloid plaques and neurofibrillary tangles. The neurotoxicity of Aβ ultimately leads to neuronal death, brain inflammation, dementia and AD. 4 Consequently, inhibition of memapsin 2 has emerged as an excellent therapeutic target for the intervention of AD. On the basis of initial kinetics and substrate specificity information, we designed a number of potent inhibitors incorporating a nonhydrolyzable Leu-Ala hydroxyethylene dipeptide isostere. 5, 6 One of the early key inhibitors was OM99-2 (1 , Figure 1). It has shown potent inhibitory activity (K i = 1.6 nM) for human memapsin 2. 6a An X-ray crystal structure of 1-bound memapsin 2 provided critical molecular insight and drug-design templates for the memapsin 2 active site. 7 Based upon the crystallographic information and preliminary structure-activity relationship studies, we then designed a number of highly potent and selective inhibitors. 8 Inhibitor 2 incorporating Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. methylsulfonyl alanine as the P 1 -ligand and oxazolylmethyl urethane as the P 3 -ligand exhibited excellent potency (K i = 0.12 nM against memapsin 2) and selectivity over BACE-2 (3800-fold) and cathepsin D (2500-fold). 8 However, its cellular inhibitory potency was in the low micromolar range (IC 50 of 1.4 μM) in Chinese hamster ovary cells. NIH Public AccessTo further improve inhibitor properties, we subsequently developed inhibitors incorporating substituted isophthalamides as P 2 -P 3 ligands in combination with the Leu-Ala hydroxyethylene dipeptide isostere. 9 As shown in Figure 1, inh...

Structure-based design, synthesis and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1′-ligands. These ligands are designed to interact with Gly-27′ carbonyl and Arg-8 side chain in the S1′-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with an (S)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. Inhibitor 19b maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1′-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1' and S2-subsites of HIV-1 protease.

Staphylococcus epidermidis and Staphylococcus aureus are important human pathogens responsible for two-thirds of all postsurgical infections of indwelling medical devices. Staphylococci form robust biofilms that provide a reservoir for chronic infection, and antibioticresistant isolates are increasingly common in both healthcare and community settings. Novel treatments that can simultaneously inhibit biofilm formation and antibiotic-resistance pathways are urgently needed to combat the increasing rates of antibiotic-resistant infections. Herein we report that loratadine, an FDA-approved antihistamine, significantly inhibits biofilm formation in both S. aureus and S. epidermidis. Furthermore, loratadine potentiates β-lactam antibiotics in methicillin-resistant strains of S. aureus and potentiates both β-lactam antibiotics and vancomycin in vancomycin-resistant strains of S. aureus. Additionally, we elucidate loratadine's mechanism of action as a novel inhibitor of the regulatory PASTA kinases Stk and Stk1 in S. epidermidis and S. aureus, respectively. Finally, we describe how Stk1 inhibition affects the expression of genes involved in both biofilm formation and antibiotic resistance in S. epidermidis and S. aureus.

The purpose of this study was to develop and optimize different processing, extraction, amplification, and sequencing methods for metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) specimens. We applied mNGS to 10 CSF samples with known standard-of-care testing (SoC) results (8 positive and 2 negative). Each sample was subjected to nine different methods by varying the sample processing protocols (supernatant, pellet, neat CSF), sample pretreatment (with or without bead beating), and the requirement of nucleic acid amplification steps using DNA sequencing (DNASeq) (with or without whole-genome amplification [WGA]) and RNA sequencing (RNASeq) methods. Negative extraction controls (NECs) were used for each method variation (4/CSF sample). Host depletion (HD) was performed on a subset of samples. We correctly determined the pathogen in 7 of 8 positive samples by mNGS compared to SoC. The two negative samples were correctly interpreted as negative. The processing protocol applied to neat CSF specimens was found to be the most successful technique for all pathogen types. While bead beating introduced bias, we found it increased the detection yield of certain organism groups. WGA prior to DNASeq was beneficial for defining pathogens at the positive threshold, and a combined DNA and RNA approach yielded results with a higher confidence when detected by both methods. HD was required for detection of a low-level-positive enterovirus sample. We demonstrate that NECs are required for interpretation of these complex results and that it is important to understand the common contaminants introduced during mNGS. Optimizing mNGS requires the use of a combination of techniques to achieve the most sensitive, agnostic approach that nonetheless may be less sensitive than SoC tools.

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The prevalence of fungi in the respiratory tracts of cystic fibrosis (CF) patients has risen. However, fungal surveillance is not routinely performed in most clinical centers in the United States, which may lead to an underestimation of the true prevalence of the problem. We conducted a prospective study comparing the rates of detection for clinically important fungi (CIF), defined as ,, and species and, in CF sputa using standard bacterial and selective fungal culture media, including Sabouraud dextrose agar with gentamicin (SDA), inhibitory mold agar (IMA), and brain heart infusion (BHI) agar with chloramphenicol and gentamicin. We described the prevalence of these fungi in an adult CF population. A total of 487 CF respiratory samples were collected from 211 unique participants. CIF were detected in 184 (37.8%) samples. Only 26.1% of CIF-positive samples were detected in bacterial culture medium, whereas greater rates of detection for fungi were found in IMA (65.8%; < 0.001), in SDA (at 30°C, 64.7%; = 0.005), and in BHI agar (63.0%; = 0.001). The prevalences of and species were 40.8% and 5.2%, respectively, which are greater than the nationally reported prevalence numbers of 20.4% and 1.9%. Selective fungal culture media and longer incubation periods yielded higher rates of detection for CIF in CF sputum samples compared with that detected in bacterial culture medium, resulting in an underdetection of fungi by bacterial culture alone. The prevalence of fungi in CF may be better estimated by using selective fungal culture media, and this may translate to important clinical decisions.

Next-generation sequencing (NGS) workflows applied to bronchoalveolar lavage (BAL) fluid specimens could enhance the detection of respiratory pathogens, although optimal approaches are not defined. This study evaluated the performance of the Respiratory Pathogen ID/AMR (RPIP) kit (Illumina, Inc.) with automated Explify bioinformatic analysis (IDbyDNA, Inc.), a targeted NGS workflow enriching specific pathogen sequences and antimicrobial resistance (AMR) markers, and a complementary untargeted metagenomic workflow with in-house bioinformatic analysis.