scholarly journals Solution NMR readily reveals distinct structural folds and interactions in doubly 13C- and 19F-labeled RNAs

2020 ◽  
Vol 6 (41) ◽  
pp. eabc6572
Author(s):  
Owen B. Becette ◽  
Guanghui Zong ◽  
Bin Chen ◽  
Kehinde M. Taiwo ◽  
David A. Case ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Sensitivity ◽  
Binding Pocket ◽  
In Vitro Transcription ◽  
Rapid Identification ◽  
Spin Pair ◽  
Encapsidation Signal ◽  
Spin Pairs ◽  
Critical Components

RNAs form critical components of biological processes implicated in human diseases, making them attractive for small-molecule therapeutics. Expanding the sites accessible to nuclear magnetic resonance (NMR) spectroscopy will provide atomic-level insights into RNA interactions. Here, we present an efficient strategy to introduce 19F-13C spin pairs into RNA by using a 5-fluorouridine-5′-triphosphate and T7 RNA polymerase–based in vitro transcription. Incorporating the 19F-13C label in two model RNAs produces linewidths that are twice as sharp as the commonly used 1H-13C spin pair. Furthermore, the high sensitivity of the 19F nucleus allows for clear delineation of helical and nonhelical regions as well as GU wobble and Watson-Crick base pairs. Last, the 19F-13C label enables rapid identification of a small-molecule binding pocket within human hepatitis B virus encapsidation signal epsilon (hHBV ε) RNA. We anticipate that the methods described herein will expand the size limitations of RNA NMR and aid with RNA-drug discovery efforts.

scholarly journals Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor

2014 ◽  
Vol 89 (1) ◽  
pp. 208-219 ◽  
Author(s):  
Jiong Shi ◽  
Jing Zhou ◽  
Upul D. Halambage ◽  
Vaibhav B. Shah ◽  
Mallori J. Burse ◽  
...  
Keyword(s):  
Amino Acid ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Binding Pocket ◽  
Host Protein ◽  
Viral Capsid ◽  
Inhibitory Protein ◽  
Hiv 1 ◽  
Ca Protein

ABSTRACTThe HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexesin vitroand permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes.IMPORTANCEThe HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.

scholarly journals A direct fluorometric activity assay for lipid kinases and phosphatases

2020 ◽  
Vol 61 (6) ◽  
pp. 945-952
Author(s):  
Jiachen Sun ◽  
Indira Singaram ◽  
Mona Hoseini Soflaee ◽  
Wonhwa Cho
Keyword(s):  
Real Time ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Modular Design ◽  
Acyl Chain ◽  
High Sensitivity ◽  
Activity Assay ◽  
Lipid Species ◽  
Lipid Kinases

Lipid kinases and phosphatases play key roles in cell signaling and regulation, are implicated in many human diseases, and are thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here, we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Because of its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided detailed mechanistic information about the product inhibition and substrate acyl-chain selectivity of PI3K and enabled rapid evaluation of small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.

scholarly journals Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene.

1994 ◽  
Vol 14 (9) ◽  
pp. 6398-6408 ◽  
Author(s):  
K H O'Donnell ◽  
C T Chen ◽  
P C Wensink
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Stages ◽  
Germ Line ◽  
Core Promoter ◽  
High Sensitivity ◽  
In Vitro Transcription ◽  
Gene Products ◽  
Position Effects ◽  
Germ Line Transformation

We identify DNA regions that are necessary for the ubiquitous expression of the Drosophila melanogaster alpha 1-tubulin (alpha 1t) gene. In vitro transcription showed that two upstream regions, tubulin element 1 (TE1 [29 bp]) and tubulin element 2 (TE2 [68 bp]), and a downstream region activate transcription. Germ line transformation demonstrated that these three regions are sufficient to direct the alpha 1t core promoter to begin transcribing at the stage of cellular blastoderm formation and to continue thereafter at high levels in all tissues and developmental stages. Remarkably, mutation of any one of these regions results in high sensitivity to chromosomal position effects, producing different but reproducible tissue-specific patterns of expression in each transformed line. None of these regions behaves as an enhancer in a conventional germ line transformation test. These observations show that these three regions, two of which bind the GAGA transcription factor, act ubiquitously to insulate from position effects and to activate transcription. The results also provide vectors for ubiquitous expression of gene products and for examining silencer activities.

scholarly journals Small-Molecule Effectors of Hepatitis B Virus Capsid Assembly Give Insight into Virus Life Cycle

2008 ◽  
Vol 82 (20) ◽  
pp. 10262-10270 ◽  
Author(s):  
Christina Bourne ◽  
Sejin Lee ◽  
Bollu Venkataiah ◽  
Angela Lee ◽  
Brent Korba ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Small Molecule ◽  
Self Assembly ◽  
Binding Pocket ◽  
Hbv Replication ◽  
B Virus ◽  
In Vitro Assembly ◽  
Kinetics Of

ABSTRACT The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors.

scholarly journals Regulation of MALAT1 triple helix stability and in vitro degradation by diphenylfurans

2020 ◽  
Vol 48 (14) ◽  
pp. 7653-7664 ◽  
Author(s):  
Anita Donlic ◽  
Martina Zafferani ◽  
Giacomo Padroni ◽  
Malavika Puri ◽  
Amanda E Hargrove
Keyword(s):  
Small Molecule ◽  
Triple Helix ◽  
Thermal Stabilization ◽  
Binding Pocket ◽  
Binding Mode ◽  
Docking Studies ◽  
In Vitro Degradation ◽  
Stem Loop ◽  
In Silico Studies

Abstract Small molecule-based modulation of a triple helix in the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been proposed as an attractive avenue for cancer treatment and a model system for understanding small molecule:RNA recognition. To elucidate fundamental recognition principles and structure–function relationships, we designed and synthesized nine novel analogs of a diphenylfuran-based small molecule DPFp8, a previously identified lead binder of MALAT1. We investigated the role of recognition modalities in binding and in silico studies along with the relationship between affinity, stability and in vitro enzymatic degradation of the triple helix. Specifically, molecular docking studies identified patterns driving affinity and selectivity, including limited ligand flexibility, as observed by ligand preorganization and 3D shape complementarity for the binding pocket. The use of differential scanning fluorimetry allowed rapid evaluation of ligand-induced thermal stabilization of the triple helix, which correlated with decreased in vitro degradation of this structure by the RNase R exonuclease. The magnitude of stabilization was related to binding mode and selectivity between the triple helix and its precursor stem loop structure. Together, this work demonstrates the value of scaffold-based libraries in revealing recognition principles and of raising broadly applicable strategies, including functional assays, for small molecule–RNA targeting.

scholarly journals Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5

2012 ◽  
Vol 449 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Guillermo Senisterra ◽  
Hong Wu ◽  
Abdellah Allali-Hassani ◽  
Gregory A. Wasney ◽  
Dalia Barsyte-Lovejoy ◽  
...  
Keyword(s):  
Small Molecule ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Protein Protein Interaction ◽  
Small Molecule Inhibition ◽  
Histone 3 ◽  
Mixed Lineage Leukaemia ◽  
Biophysical Analysis ◽  
Lower Protein

WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3–9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein–protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.

scholarly journals Structural basis of peptidomimetic agonism revealed by small molecule GLP-1R agonists Boc5 and WB4-24

2022 ◽  
Author(s):  
Zhaotong Cong ◽  
Qingtong Zhou ◽  
Yang Li ◽  
Li-Nan Chen ◽  
Zi-Chen Zhang ◽  
...  
Keyword(s):  
Small Molecule ◽  
Binding Pocket ◽  
Binding Mode ◽  
Fine Tuning ◽  
Structural Basis ◽  
Structural Framework ◽  
Biased Signaling ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of them are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric non-peptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryo-electron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3 and 7, one arm of both compounds inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8-D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts showing an interaction feature substantially similar to a previously known small molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by non-peptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine tuning of pharmacological efficacy in the development of future small molecule therapeutics targeting GLP-1R.

Insulating DNA directs ubiquitous transcription of the Drosophila melanogaster alpha 1-tubulin gene

1994 ◽  
Vol 14 (9) ◽  
pp. 6398-6408
Author(s):  
K H O'Donnell ◽  
C T Chen ◽  
P C Wensink
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Stages ◽  
Germ Line ◽  
Core Promoter ◽  
High Sensitivity ◽  
In Vitro Transcription ◽  
Gene Products ◽  
Position Effects ◽  
Germ Line Transformation

We identify DNA regions that are necessary for the ubiquitous expression of the Drosophila melanogaster alpha 1-tubulin (alpha 1t) gene. In vitro transcription showed that two upstream regions, tubulin element 1 (TE1 [29 bp]) and tubulin element 2 (TE2 [68 bp]), and a downstream region activate transcription. Germ line transformation demonstrated that these three regions are sufficient to direct the alpha 1t core promoter to begin transcribing at the stage of cellular blastoderm formation and to continue thereafter at high levels in all tissues and developmental stages. Remarkably, mutation of any one of these regions results in high sensitivity to chromosomal position effects, producing different but reproducible tissue-specific patterns of expression in each transformed line. None of these regions behaves as an enhancer in a conventional germ line transformation test. These observations show that these three regions, two of which bind the GAGA transcription factor, act ubiquitously to insulate from position effects and to activate transcription. The results also provide vectors for ubiquitous expression of gene products and for examining silencer activities.

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