ABSTRACT
The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.
Subject(s)
Artemisinins , Malaria, Falciparum , Peptide Nucleic Acids , Humans , Plasmodium falciparum/genetics , Streptavidin , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Protozoan Proteins/pharmacology , Artemisinins/pharmacology , Artemisinins/therapeutic use , Malaria, Falciparum/drug therapy , Drug Resistance/genetics , RNAABSTRACT
Coronary atherosclerosis leading to ischemic artery disease is one of the etiological factors to develop heart failure (HF). This study aimed to investigate potential biomarkers for discriminating HF in atherosclerotic patients. This study included 40 consecutive atherosclerotic patients who underwent angiography. Concentrations of B-type natriuretic peptide (BNP), fibronectin type III domain containing 5 (FNDC5), and Phosphodiesterase 9A (PDE9A) were measured in 20 atherosclerotic patients with HF symptoms/signs and 20 without HF symptoms/signs. Circulating BNP levels were elevated, while FNDC5 levels were reduced in atherosclerotic patients with HF symptoms/signs compared to those without HF symptoms/signs. Pearson correlation analysis showed a significant correlation between FNDC5 and BNP. Receiver Operating Characteristics analysis indicated that both FNDC5 and BNP were able to discriminate HF in atherosclerotic patients. Our findings suggest that FNDC5, along with BNP, has independent value as a biomarker for discriminating HF in patients with coronary atherosclerosis.
ABSTRACT
Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.
Subject(s)
Cyclopentanes , Ovarian Neoplasms , Peptide Nucleic Acids , RNA, Long Noncoding , Humans , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Female , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Peptide Nucleic Acids/chemistry , Cyclopentanes/chemistry , Cyclopentanes/pharmacology , Cell Line, Tumor , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolismABSTRACT
MERTK and AXL are members of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases that are aberrantly expressed and have been implicated as therapeutic targets in a wide variety of human tumors. Dual MERTK and AXL inhibition could provide antitumor action mediated by both direct tumor cell killing and modulation of the innate immune response in some tumors such as nonsmall cell lung cancer. We utilized our knowledge of MERTK inhibitors and a structure-based drug design approach to discover a novel class of macrocyclic dual MERTK/AXL inhibitors. The lead compound 43 had low-nanomolar activity against both MERTK and AXL and good selectivity over TYRO3 and FLT3. Its target engagement and selectivity were also confirmed by NanoBRET and cell-based MERTK and AXL phosphorylation assays. Compound 43 had excellent pharmacokinetic properties (large AUC and long half-life) and mediated antitumor activity against lung cancer cell lines, indicating its potential as a therapeutic agent.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , c-Mer Tyrosine Kinase/metabolism , Axl Receptor Tyrosine Kinase , Proto-Oncogene Proteins/metabolism , Lung Neoplasms/drug therapy , Cell Line, TumorABSTRACT
Partial endothelial-to-mesenchymal transition (EndMT) is an intermediate phenotype observed in endothelial cells (ECs) undergoing a transition toward a mesenchymal state to support neovascularization during (patho)physiological angiogenesis. Here, we investigated the occurrence of partial EndMT in ECs under hypoxic/ischemic conditions and identified general transcription factor IIH subunit 4 (GTF2H4) as a positive regulator of this process. In addition, we discovered that GTF2H4 collaborates with its target protein excision repair cross-complementation group 3 (ERCC3) to co-regulate partial EndMT. Furthermore, by using phosphorylation proteomics and site-directed mutagenesis, we demonstrated that GTF2H4 was involved in the phosphorylation of receptor coactivator 3 (NCOA3) at serine 1330, which promoted the interaction between NCOA3 and p65, resulting in the transcriptional activation of NF-κB and the NF-κB/Snail signaling axis during partial EndMT. In vivo experiments confirmed that GTF2H4 significantly promoted partial EndMT and angiogenesis after ischemic injury. Collectively, our findings reveal that targeting GTF2H4 is promising for tissue repair and offers potential opportunities for treating hypoxic/ischemic diseases.