scholarly journals Basis for drug selectivity of plasmepsin IX and X inhibition for Plasmodium falciparum and vivax

2021 ◽  
Author(s):  
Alan Cowman ◽  
Anthony Hodder ◽  
Janni Christensen ◽  
Stephen Scally ◽  
Tony Triglia ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Crystal Structures ◽  
Drug Targets ◽  
Active Sites ◽  
Drug Binding ◽  
Dual Inhibitor ◽  
3 Dimensional ◽  
Protein Substrates ◽  
Binding Cleft ◽  
Parasite Egress

Abstract Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.

scholarly journals Basis for drug selectivity of plasmepsin IX and X inhibition for Plasmodium falciparum and vivax

2021 ◽  
Author(s):  
Anthony N. Hodder ◽  
Stephen Scally ◽  
Tony Triglia ◽  
Anna Ngo ◽  
Richard W. Birkinshaw ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Crystal Structures ◽  
Drug Targets ◽  
Active Sites ◽  
Drug Binding ◽  
Dual Inhibitor ◽  
3 Dimensional ◽  
Protein Substrates ◽  
Binding Cleft ◽  
Parasite Egress

Abstract Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.

scholarly journals Discovery of a dual inhibitor of NQO1 and GSTP1 for treating glioblastoma

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Kecheng Lei ◽  
Xiaoxia Gu ◽  
Alvaro G. Alvarado ◽  
Yuhong Du ◽  
Shilin Luo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Crystal Structures ◽  
Active Sites ◽  
Redox Homeostasis ◽  
Growth Factor Receptor ◽  
Quinone Oxidoreductase ◽  
Cancer Proliferation ◽  
Dual Inhibitor

Abstract Background Glioblastoma (GBM) is a universally lethal tumor with frequently overexpressed or mutated epidermal growth factor receptor (EGFR). NADPH quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) are commonly upregulated in GBM. NQO1 and GSTP1 decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and promotes GBM cell proliferation. Methods High-throughput screen was used for agents selectively active against GBM cells with EGFRvIII mutations. Co-crystal structures were revealed molecular details of target recognition. Pharmacological and gene knockdown/overexpression approaches were used to investigate the oxidative stress in vitro and in vivo. Results We identified a small molecular inhibitor, “MNPC,” that binds to both NQO1 and GSTP1 with high affinity and selectivity. MNPC inhibits NQO1 and GSTP1 enzymes and induces apoptosis in GBM, specifically inhibiting the growth of cell lines and primary GBM bearing the EGFRvIII mutation. Co-crystal structures between MNPC and NQO1, and molecular docking of MNPC with GSTP1 reveal that it binds the active sites and acts as a potent dual inhibitor. Inactivation of both NQO1 and GSTP1 with siRNA or MNPC results in imbalanced redox homeostasis, leading to apoptosis and mitigated cancer proliferation in vitro and in vivo. Conclusions Thus, MNPC, a dual inhibitor for both NQO1 and GSTP1, provides a novel lead compound for treating GBM via the exploitation of specific vulnerabilities created by mutant EGFR.

scholarly journals Dynamics of human protein kinase Aurora A linked to drug selectivity

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Warintra Pitsawong ◽  
Vanessa Buosi ◽  
Renee Otten ◽  
Roman V Agafonov ◽  
Adelajda Zorba ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Kinase Inhibitors ◽  
Bound State ◽  
Drug Binding ◽  
Aurora A ◽  
Drug Selectivity ◽  
Approved Drugs

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.

scholarly journals Dynamics of human protein kinases linked to drug selectivity

2018 ◽  
Author(s):  
Warintra Pitsawong ◽  
Vanessa Buosi ◽  
Renee Otten ◽  
Roman V. Agafonov ◽  
Adelajda Zorba ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Kinases ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Kinase Inhibitors ◽  
Drug Binding ◽  
Inhibition Mechanism ◽  
Aurora A ◽  
Drug Selectivity

AbstractProtein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.eLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

scholarly journals Crystal structures of human MGST2 reveal synchronized conformational changes regulating catalysis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Madhuranayaki Thulasingam ◽  
Laura Orellana ◽  
Emmanuel Nji ◽  
Shabbir Ahmad ◽  
Agnes Rinaldo-Matthis ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Oxidative Dna Damage ◽  
Glutathione S Transferase ◽  
Concerted Mechanism ◽  
Related Drug ◽  
Stress Oxidative ◽  
And Control

AbstractMicrosomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets.

scholarly journals Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds

2019 ◽  
Author(s):  
Madeline G. Dans ◽  
Greta E. Weiss ◽  
Danny W. Wilson ◽  
Brad E. Sleebs ◽  
Brendan S. Crabb ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasmodium Falciparum ◽  
Drug Targets ◽  
Neglected Tropical Diseases ◽  
Antimalarial Activity ◽  
Specific Inhibitor ◽  
Substantial Effect ◽  
Blood Stage ◽  
Asexual Blood Stage ◽  
Parasite Egress

AbstractTo identify potential inhibitors of egress and invasion in the asexual blood stage of Plasmodium falciparum, we screened the Medicines for Malaria Venture (MMV) Pathogen Box. This compound library comprises of 400 drugs against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, Nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has greater implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.ImportancePlasmodium falciparum causes the most severe form of malaria and with emerging resistance to frontline treatments, there is the need to identify new drug targets in the parasite. One of the most critical processes during the asexual blood stage in the parasite’s lifecycle is the egress from old red blood cells (RBCs) and subsequent invasion of new RBCs. Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. Identifying novel compounds that inhibit these essential processes would further their development into possible antimalarials that would be highly effective at killing asexual RBC stage parasites when used in combination with drugs that target the intraerythrocytic growth phase. These compounds potentially may also be used as novel tools to study the complex biology of parasites to gain further insight into the mechanisms behind egress and invasion.

Antimalarial Drug Targets and Drugs Targeting Dolichol Metabolic Pathway of Plasmodium falciparum

2014 ◽  
Vol 15 (4) ◽  
pp. 374-409 ◽  
Author(s):  
Tabish Qidwai ◽  
Avantika Priya ◽  
Nihal Khan ◽  
Himanshu Tripathi ◽  
Feroz Khan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Metabolic Pathway ◽  
Antimalarial Drug ◽  
Drug Targets

scholarly journals DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions

2021 ◽  
Author(s):  
Anirban Ghosh ◽  
Eric Largy ◽  
Valérie Gabelica
Keyword(s):  
Mass Spectrometry ◽  
Drug Targets ◽  
Native Mass Spectrometry ◽  
Drug Binding ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
Nmr Structures ◽  
G Quadruplex ◽  
Screening Assays

Abstract G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

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