Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC 50 s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC 50 s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Dengue viruses (DENVs) are enveloped positive-strand RNA viruses and belong to the family Flaviviridae. DENV is the most important arthropod-borne viral infection. Over one-third of the world population lives in areas of DENV endemicity, and an estimated 390 million infections occur every year. In addition, the number of countries having experienced DENV epidemics has risen from 9 in 1970 to more than 100 today (1, 2). Furthermore, the number of diagnosed infections across America, Southeast Asia, and the Western Pacific nearly doubled from 1.2 million in 2008 to over 2.3 million in 2010 (2). Four different DENV serotypes have been identified so far. Recently, evidence for an additional subtype has been presented (3). Serotypes 1 to 4 are now prevalent in Asia, Africa, and America, and the regions where dengue is endemic are still increasing (4-6), with dengue endangering even Europe and the United States due to vector spread. DENV infections can be associated with dengue fever, but up to 88% of the infections remain inapparent (7). These nonpersistent infected patients serve besides persistently infected mosquitoes as a virus reservoir. Severe DENV infections and especially reinfections may lead to dengue hemorrhagic fever and dengue shock syndrome, with lethality up to 5% (2,8,9). There is neither a vaccination nor a specific treatment for DENV infections.The DENV genome contains a single open reading frame, which encodes the structural proteins capsid, membrane precursor (prM), and envelope and the nonstructural proteins NS1, NS2, NS3, NS4, and NS5 (10). Cellular proteases and the viral serine protease (PR) are responsible for cleaving the viral precursor polyprotein into functional proteins. The DENV PR consists of the amino-terminal domain of the NS3 protein and requires NS2B, a 14-kDa protein, as a cofactor to form a stable complex. This heterodimeric PR cleaves at the capsid-prM, NS2A/NS2...
Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.
The nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor associated with macrophage polarization. However, its function in tumor-associated macrophages (TAMs) has not been investigated to date. Here, we report the PPARβ/δ-regulated transcriptome and cistrome for TAMs from ovarian carcinoma patients. Comparison with monocyte-derived macrophages shows that the vast majority of direct PPARβ/δ target genes are upregulated in TAMs and largely refractory to synthetic agonists, but repressible by inverse agonists. Besides genes with metabolic functions, these include cell type-selective genes associated with immune regulation and tumor progression, e.g., LRP5, CD300A, MAP3K8 and ANGPTL4. This deregulation is not due to increased expression of PPARβ/δ or its enhanced recruitment to target genes. Instead, lipidomic analysis of malignancy-associated ascites revealed high concentrations of polyunsaturated fatty acids, in particular linoleic acid, acting as potent PPARβ/δ agonists in macrophages. These fatty acid ligands accumulate in lipid droplets in TAMs, thereby providing a reservoir of PPARβ/δ ligands. These observations suggest that the deregulation of PPARβ/δ target genes by ligands of the tumor microenvironment contributes to the pro-tumorigenic polarization of ovarian carcinoma TAMs. This conclusion is supported by the association of high ANGPTL4 expression with a shorter relapse-free survival in serous ovarian carcinoma.
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