Annette Evangelisti | University of New Mexico (original) (raw)
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Papers by Annette Evangelisti
Journal of biomolecular screening, Jan 6, 2015
A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to tr... more A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluor...
Drug Discovery Today: Technologies, 2014
This review highlights the concepts, recent applications and limitations of High Throughput Scree... more This review highlights the concepts, recent applications and limitations of High Throughput Screening (HTS) flow cytometry-based efflux inhibitory assays. This platform has been employed in mammalian and yeast efflux systems leading to the identification of small molecules with transporter inhibitory capabilities. This technology offers the possibility of substrate multiplexing and may promote novel strategies targeting microbial efflux systems. This platform can generate a comprehensive dataset that may support efforts to map the interface between chemistry and transporter biology in a variety of pathogenic systems.
Although many mechanisms exist, resistance of tumors to cancer therapy drugs is the principal rea... more Although many mechanisms exist, resistance of tumors to cancer therapy drugs is the principal reason for treatment failure and the majority of clinical and experimental data indicates that multidrug transporters such ABCB1 (a.k.a. Pgp, MDR1) and ABCG2 (a.k.a. BCRP, MRP1) play a leading role by preventing cytotoxic intracellular drug concentrations. Inhibition of the function of these drug efflux pumps presents a promising approach to treat cancer using existing drugs. To date, clinical trials with such adjuvant therapies have been relatively unsuccessful. One likely contributing factor to these failed clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We propose that searching for selective efflux inhibitors by profiling multiple ABC transporter efflux pumps against a library of small molecules could result in molecular probes that could further explore such interactions. Using the mitochondrial membrane potential dye JC-1 as a dual-pump fluorescent reporter substrate in our primary screening protocol we observed a piperazine substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure activity relationship (SAR) driven chemistry effort we describe herein a selective ABCG2 efflux inhibitor (SID 88095709) with a 36 fold preference over ABCB1 with increased activity over prior art for ABCG2. The probe has low in vitro cellular toxicity, as well as adequate solubility and stability under appropriate experimental conditions. The probe also appears to have an IP landscape with space to operate. In vitro chemotherapeutic potentiation further illustrates the utility of the probe compound and related members. A related scaffold (SID 97301789) also shows promise for further development and optimization.
Analytical Biochemistry, 2013
L.A. Sklar). 1 These authors contributed equally to this work. 2 Abbreviations used: ABC, ATP bin... more L.A. Sklar). 1 These authors contributed equally to this work. 2 Abbreviations used: ABC, ATP binding cassette; P-gp, P-glycoprotein (ABCB1); MRP1, multidrug resistance protein 1 (ABCC1); BCRP, breast cancer resistance protein (ABCG2); JC-1, 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolcarbocyanine iodide; CaAM, calcein acetoxymethyl ester; DMSO, dimethyl sulfoxide; Vin, vincristine; PBS, phosphate-buffered saline; MCF, median channel fluorescence; HTS, highthroughput screening; MDR, multidrug resistance.
Journal of Experimental Zoology …, Jan 1, 2004
We analyze the structure of the yeast transcriptional regulation network, as revealed by chromati... more We analyze the structure of the yeast transcriptional regulation network, as revealed by chromatin immunoprecipitation experiments, and characterize the molecular evolution of both its transcriptional regulators and their target (regulated) genes. We test the hypothesis that highly connected genes are more important to the function of gene networks. Three lines of evidence, the rate of molecular evolution of network genes, the rate at which network genes undergo gene duplication, and the effects of synthetic null mutation in network genes provide no strong support for this hypothesis. In addition, we ask how network genes diverge in their transcriptional regulation after duplication. Both loss (subfunctionalization) and gain (neofunctionalization) of transcription factor binding play a role in this divergence, which is often rapid. On one hand, gene duplicates experience a net loss in the number of transcription factors binding to them, indicating the importance of losing transcription factor binding sites after gene duplication. On the other hand, the number of transcription factors that bind to highly diverged duplicates is significantly greater than expected if loss of binding played the only role in the divergence of duplicate genes.
Genome biology and evolution, Jan 1, 2010
By comparing the patterns of evolution in the coding and upstream noncoding regions of yeast ribo... more By comparing the patterns of evolution in the coding and upstream noncoding regions of yeast ribosomal protein (RP) genes duplicated in a genome duplication, we find that although nonsynonymous sites in the coding sequences show strong evidence for the fixation of recent gene conversion events, similar patterns are less evident among the synonymous positions and noncoding regulatory elements. This result suggests a potential explanation for the somewhat puzzling fact that duplicated RP genes are not functionally redundant despite their very high protein sequence identity. An analysis of the patterns of regulatory network evolution after genome duplication also indicates that the duplicated proteins have diverged considerably in expression despite their similar protein sequences.
Journal of biomolecular screening, Jan 6, 2015
A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to tr... more A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluor...
Drug Discovery Today: Technologies, 2014
This review highlights the concepts, recent applications and limitations of High Throughput Scree... more This review highlights the concepts, recent applications and limitations of High Throughput Screening (HTS) flow cytometry-based efflux inhibitory assays. This platform has been employed in mammalian and yeast efflux systems leading to the identification of small molecules with transporter inhibitory capabilities. This technology offers the possibility of substrate multiplexing and may promote novel strategies targeting microbial efflux systems. This platform can generate a comprehensive dataset that may support efforts to map the interface between chemistry and transporter biology in a variety of pathogenic systems.
Although many mechanisms exist, resistance of tumors to cancer therapy drugs is the principal rea... more Although many mechanisms exist, resistance of tumors to cancer therapy drugs is the principal reason for treatment failure and the majority of clinical and experimental data indicates that multidrug transporters such ABCB1 (a.k.a. Pgp, MDR1) and ABCG2 (a.k.a. BCRP, MRP1) play a leading role by preventing cytotoxic intracellular drug concentrations. Inhibition of the function of these drug efflux pumps presents a promising approach to treat cancer using existing drugs. To date, clinical trials with such adjuvant therapies have been relatively unsuccessful. One likely contributing factor to these failed clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We propose that searching for selective efflux inhibitors by profiling multiple ABC transporter efflux pumps against a library of small molecules could result in molecular probes that could further explore such interactions. Using the mitochondrial membrane potential dye JC-1 as a dual-pump fluorescent reporter substrate in our primary screening protocol we observed a piperazine substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure activity relationship (SAR) driven chemistry effort we describe herein a selective ABCG2 efflux inhibitor (SID 88095709) with a 36 fold preference over ABCB1 with increased activity over prior art for ABCG2. The probe has low in vitro cellular toxicity, as well as adequate solubility and stability under appropriate experimental conditions. The probe also appears to have an IP landscape with space to operate. In vitro chemotherapeutic potentiation further illustrates the utility of the probe compound and related members. A related scaffold (SID 97301789) also shows promise for further development and optimization.
Analytical Biochemistry, 2013
L.A. Sklar). 1 These authors contributed equally to this work. 2 Abbreviations used: ABC, ATP bin... more L.A. Sklar). 1 These authors contributed equally to this work. 2 Abbreviations used: ABC, ATP binding cassette; P-gp, P-glycoprotein (ABCB1); MRP1, multidrug resistance protein 1 (ABCC1); BCRP, breast cancer resistance protein (ABCG2); JC-1, 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolcarbocyanine iodide; CaAM, calcein acetoxymethyl ester; DMSO, dimethyl sulfoxide; Vin, vincristine; PBS, phosphate-buffered saline; MCF, median channel fluorescence; HTS, highthroughput screening; MDR, multidrug resistance.
Journal of Experimental Zoology …, Jan 1, 2004
We analyze the structure of the yeast transcriptional regulation network, as revealed by chromati... more We analyze the structure of the yeast transcriptional regulation network, as revealed by chromatin immunoprecipitation experiments, and characterize the molecular evolution of both its transcriptional regulators and their target (regulated) genes. We test the hypothesis that highly connected genes are more important to the function of gene networks. Three lines of evidence, the rate of molecular evolution of network genes, the rate at which network genes undergo gene duplication, and the effects of synthetic null mutation in network genes provide no strong support for this hypothesis. In addition, we ask how network genes diverge in their transcriptional regulation after duplication. Both loss (subfunctionalization) and gain (neofunctionalization) of transcription factor binding play a role in this divergence, which is often rapid. On one hand, gene duplicates experience a net loss in the number of transcription factors binding to them, indicating the importance of losing transcription factor binding sites after gene duplication. On the other hand, the number of transcription factors that bind to highly diverged duplicates is significantly greater than expected if loss of binding played the only role in the divergence of duplicate genes.
Genome biology and evolution, Jan 1, 2010
By comparing the patterns of evolution in the coding and upstream noncoding regions of yeast ribo... more By comparing the patterns of evolution in the coding and upstream noncoding regions of yeast ribosomal protein (RP) genes duplicated in a genome duplication, we find that although nonsynonymous sites in the coding sequences show strong evidence for the fixation of recent gene conversion events, similar patterns are less evident among the synonymous positions and noncoding regulatory elements. This result suggests a potential explanation for the somewhat puzzling fact that duplicated RP genes are not functionally redundant despite their very high protein sequence identity. An analysis of the patterns of regulatory network evolution after genome duplication also indicates that the duplicated proteins have diverged considerably in expression despite their similar protein sequences.