scholarly journals Identification of a ligand binding hot spot and structural motifs replicating aspects of tyrosyl-DNA phosphodiesterase I (TDP1) phosphoryl recognition by crystallographic fragment cocktail screening

2019 ◽  
Vol 47 (19) ◽  
pp. 10134-10150 ◽  
Author(s):  
George T Lountos ◽  
Xue Zhi Zhao ◽  
Evgeny Kiselev ◽  
Joseph E Tropea ◽  
Danielle Needle ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Small Molecule ◽  
Anticancer Agents ◽  
Active Site ◽  
Catalytic Domain ◽  
Hot Spot ◽  
Alkylating Agents ◽  
Fragment Screening ◽  
Rational Development ◽  
Biochemical Assays

Abstract Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3′ end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.

scholarly journals Biological and Structural Characterization of Trypanosoma cruzi Phosphodiesterase C and Implications for Design of Parasite Selective Inhibitors

2012 ◽  
Vol 287 (15) ◽  
pp. 11788-11797 ◽  
Author(s):  
Huanchen Wang ◽  
Stefan Kunz ◽  
Gong Chen ◽  
Thomas Seebeck ◽  
Yiqian Wan ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Trypanosoma Evansi ◽  
Active Site Residues ◽  
Dual Specificity ◽  
Pde Inhibitors ◽  
Starting Model

Trypanosoma cruzi phosphodiesterase C (TcrPDEC) is a potential new drug target for the treatment of Chagas disease but has not been well studied. This study reports the enzymatic properties of various kinetoplastid PDECs and the crystal structures of the unliganded TcrPDEC1 catalytic domain and its complex with an inhibitor. Mutations of PDEC during the course of evolution led to inactivation of PDEC in Trypanosoma brucei/Trypanosoma evansi/Trypanosoma congolense, whereas the enzyme is active in all other kinetoplastids. The TcrPDEC1 catalytic domain hydrolyzes both cAMP and cGMP with a Km of 23.8 μm and a kcat of 31 s−1 for cAMP and a Km of 99.1 μm and a kcat of 17 s−1 for cGMP, thus confirming its dual specificity. The crystal structures show that the N-terminal fragment wraps around the TcrPDEC catalytic domain and may thus regulate its enzymatic activity via direct interactions with the active site residues. A PDE5 selective inhibitor that has an IC50 of 230 nm for TcrPDEC1 binds to TcrPDEC1 in an orientation opposite to that of sildenafil. This observation, together with the screen of the inhibitory potency of human PDE inhibitors against TcrPDEC, implies that the scaffold of some human PDE inhibitors might be used as the starting model for design of parasite PDE inhibitors. The structural study also identified a unique parasite pocket that neighbors the active site and may thus be valuable for the design of parasite-specific inhibitors.

scholarly journals Crystal structures of hFPPS in complex with novel anticancer drug leads

2014 ◽  
Vol 70 (a1) ◽  
pp. C712-C712
Author(s):  
Jaeok Park ◽  
Chun Leung ◽  
Yih-Shyan Lin ◽  
Joris De Schutter ◽  
Youla Tsantrizos ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Crystal Structures ◽  
Anticancer Agents ◽  
Active Site ◽  
Farnesyl Pyrophosphate ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Inhibitory Site ◽  
Active Site Cavity

Human farnesyl pyrophosphate synthase (hFPPS) produces farnesyl pyrophosphate, an isoprenoid required for a variety of essential cellular processes. Inhibition of hFPPS has been well established as the mechanism of action of the nitrogen-containing bisphosphonate (N-BP) drugs, currently best known for their anti-bone resorptive effects. Recent investigations indicate that hFPPS inhibition also produces potent anticancer effects both in vitro and vivo: N-BPs inhibit proliferation, motility, and viability of tumor cells, and act in synergy with other anticancer agents [1,2]. However, the physicochemical properties of the current N-BP drugs seriously compromise their full anticancer potential in non-skeletal tissues. They show poor membrane permeability and extreme affinity to bone, due mainly to their highly charged bisphosphonate moiety, which mimics the pyrophosphate of the substrates of hFPPS. Both the substrates and N-BPs bind to hFPPS via Mg ion-mediated interactions between their pyrophosphate/bisphosphonate moiety and two aspartate-rich surfaces of the enzyme's active site cavity. Recently, we took a structure-guided approach to develop bisphosphonates with higher lipophilicity for enhanced uptake into non-skeletal tissues. Surprisingly, some of the new compounds were found to bind to hFPPS even in the absence of Mg ions. Crystal structures of hFPPS in complex with a representative compound revealed that this bisphosphonate binds to the enzyme's active site in the presence of Mg ions, but also to a nearby allosteric inhibitory site in their absence. Furthermore, removal of a phosphonate group from the bisphosphonate moiety of this compound resulted in an inhibitor that binds exclusively to the allosteric site. Based on the crystal structures with these lead compounds, we generated of a novel class of non-bisphosphonate, allosteric inhibitors of hFPPS with superior physicochemical properties than those of the current N-BP drugs for broader tissue distribution.

Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

2019 ◽  
Vol 75 (12) ◽  
pp. 1138-1147
Author(s):  
Hsiao-Chuan Huang ◽  
Liu-Hong Qi ◽  
Yo-Chia Chen ◽  
Li-Chu Tsai
Keyword(s):  
Enzymatic Hydrolysis ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Sites ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Sites ◽  
Key Residues ◽  
Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

scholarly journals A Second Look at the Crystal Structures of Drosophila melanogaster Acetylcholinesterase in Complex with Tacrine Derivatives Provides Insights Concerning Catalytic Intermediates and the Design of Specific Insecticides

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1198 ◽  
Author(s):  
Florian Nachon ◽  
Terrone L. Rosenberry ◽  
Israel Silman ◽  
Joel L. Sussman
Keyword(s):  
Drosophila Melanogaster ◽  
Crystal Structures ◽  
Small Molecule ◽  
Diffraction Data ◽  
Active Site ◽  
Structure Refinement ◽  
Second Look ◽  
Density Maps ◽  
Original Analysis ◽  
Public Data

Over recent decades, crystallographic software for data processing and structure refinement has improved dramatically, resulting in more accurate and detailed crystal structures. It is, therefore, sometimes valuable to have a second look at “old” diffraction data, especially when earlier interpretation of the electron density maps was rather difficult. Here, we present updated crystal structures of Drosophila melanogaster acetylcholinesterase (DmAChE) originally published in [Harel et al., Prot Sci (2000) 9:1063-1072], which reveal features previously unnoticed. Thus, previously unmodeled density in the native active site can be interpreted as stable acetylation of the catalytic serine. Similarly, a strong density in the DmAChE/ZA complex originally attributed to a sulfate ion is better interpreted as a small molecule that is covalently bound. This small molecule can be modeled as either a propionate or a glycinate. The complex is reminiscent of the carboxylate butyrylcholinesterase complexes observed in crystal structures of human butyrylcholinesterases from various sources, and demonstrates the remarkable ability of cholinesterases to stabilize covalent complexes with carboxylates. A very strong peak of density (10 σ) at covalent distance from the Cβ of the catalytic serine is present in the DmAChE/ZAI complex. This can be undoubtedly attributed to an iodine atom, suggesting an unanticipated iodo/hydroxyl exchange between Ser238 and the inhibitor, possibly driven by the intense X-ray irradiation. Finally, the binding of tacrine-derived inhibitors, such as ZA (1DX4) or the iodinated analog, ZAI (1QON) results in the appearance of an open channel that connects the base of the active-site gorge to the solvent. This channel, which arises due to the absence of the conserved tyrosine present in vertebrate cholinesterases, could be exploited to design inhibitors specific to insect cholinesterases. The present study demonstrates that updated processing of older diffraction images, and the re-refinement of older diffraction data, can produce valuable information that could not be detected in the original analysis, and strongly supports the preservation of the diffraction images in public data banks.

A GH13 α-glucosidase from Weissella cibaria uncommonly acts on short-chain maltooligosaccharides

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Karan Wangpaiboon ◽  
Pasunee Laohawuttichai ◽  
Sun-Yong Kim ◽  
Tomoyuki Mori ◽  
Santhana Nakapong ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Hydrolytic Activity ◽  
Size Exclusion ◽  
Short Chain ◽  
Glycoside Hydrolase Family ◽  
Enzyme Active Site ◽  
Weissella Cibaria ◽  
Terminal Domain

α-Glucosidase (EC 3.2.1.20) is a carbohydrate-hydrolyzing enzyme which generally cleaves α-1,4-glycosidic bonds of oligosaccharides and starch from the nonreducing ends. In this study, the novel α-glucosidase from Weissella cibaria BBK-1 (WcAG) was biochemically and structurally characterized. WcAG belongs to glycoside hydrolase family 13 (GH13) and to the neopullanase subfamily. It exhibits distinct hydrolytic activity towards the α-1,4 linkages of short-chain oligosaccharides from the reducing end. The enzyme prefers to hydrolyse maltotriose and acarbose, while it cannot hydrolyse cyclic oligosaccharides and polysaccharides. In addition, WcAG can cleave pullulan hydrolysates and strongly exhibits transglycosylation activity in the presence of maltose. Size-exclusion chromatography and X-ray crystal structures revealed that WcAG forms a homodimer in which the N-terminal domain of one monomer is orientated in proximity to the catalytic domain of another, creating the substrate-binding groove. Crystal structures of WcAG in complexes with maltose, maltotriose and acarbose revealed a remarkable enzyme active site with accessible +2, +1 and −1 subsites, along with an Arg–Glu gate (Arg176–Glu296) in front of the active site. The −2 and −3 subsites were blocked by Met119 and Asn120 from the N-terminal domain of a different subunit, resulting in an extremely restricted substrate preference.

scholarly journals Roles of the hydroxy group of tyrosine in crystal structures of Sulfurisphaera tokodaii O 6-methylguanine-DNA methyltransferase

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Makiko Kikuchi ◽  
Takahiro Yamauchi ◽  
Yasuhito Iizuka ◽  
Masaru Tsunoda
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Hydroxy Group ◽  
Active Site ◽  
Dna Methyltransferase ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Alkylating Agents ◽  
Substrate Analogs ◽  
Methylguanine Dna Methyltransferase

O 6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O 6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13–2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O 6-methyl-2′-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix–turn–helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

scholarly journals Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

2019 ◽  
Vol 75 (10) ◽  
pp. 895-903 ◽  
Author(s):  
K. Koruza ◽  
B. Lafumat ◽  
M. Nyblom ◽  
B. P. Mahon ◽  
W. Knecht ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Crystal Structures ◽  
Active Site ◽  
Catalytic Domain ◽  
Crystal Form ◽  
Carbonic Anhydrase Ix ◽  
Structural Comparison ◽  
X Ray ◽  
Ca Ix ◽  
Human Carbonic Anhydrase

Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

Abstract 10306: Identification and Characterization of Orally Bioavailable Small Molecule Protease Proprotein Convertase Subtilisin-like Kexin Type 9 Inhibitors (Best of Basic Science Abstract)

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Nabil A Elshourbagy ◽  
Harold V Meyers ◽  
Shaker A Mousa ◽  
Sherin S Abdel-Meguid
Keyword(s):  
Small Molecule ◽  
Active Site ◽  
Catalytic Domain ◽  
Significant Advance ◽  
High Cholesterol Diet ◽  
Proprotein Convertase ◽  
Mortality And Morbidity ◽  
Autocatalytic Processing ◽  
Nanomolar Range

Intervention with drugs to reduce low density lipoprotein-cholesterol (LDL-C) has proven to decrease mortality and morbidity. Here, we report the development of small molecules that lower LDL-C by targeting the LDL-receptor (LDLR) degradation pathway, which is modulated by the protease proprotein convertase subtilisin-like kexin type 9 (PCSK9). PCSK9 is synthesized as a 72-kDa zymogen that undergoes autocatalytic processing between the prodomain and catalytic domain, an important step that is absolutely required for secretion of PCSK9 and its function. Secreted PCSK9 binds to the LDLR and enhances its degradation. We have identified inhibitors of the autocatalytic processing by virtual docking of millions of commercial compounds into the atomic structure of the PCSK9 active site. Top scoring compounds that showed the best fit in the active site were selected for screening. These compounds were tested for their ability to inhibit the autocatalytic processing/secretion of PCSK9. The most potent compounds exhibited a concentration-dependent inhibition of the PCSK9 processing/secretion with IC50’s in the nanomolar range. These compounds were tested for selectivity against other PCSK’s. The data demonstrated that these compounds were selective in that they inhibit PCSK9 processing/secretion without affecting the secretion of other related PCSK’s. Furthermore, these compounds exhibited a 5- to 10-fold LDLR upregulation at 1.6 μM in two recombinant cell-based assays as well as exhibited an increase in the fluorescently labeled DiI-LDL uptake in situ in the nanomolar range. In animal studies, intraperitoneal (IP) injection of these compounds administered alone resulted in more than 20% reduction in LDL-C in mice fed a high fat/high cholesterol diet and over 40% reduction in LDL-C if administered in combination with atorvastatin. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation of these compounds demonstrated that they are orally efficacious, with one compound exhibiting 43% oral bioavailability. Thus, identifying small molecule, orally active PCSK9 modulators represents a significant advance and opportunityfor drug development.

scholarly journals Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

2019 ◽  
Author(s):  
Ke Shi ◽  
Özlem Demir ◽  
Michael A. Carpenter ◽  
Surajit Banerjee ◽  
Daniel A. Harki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Active Site ◽  
Rational Design ◽  
Catalytic Domain ◽  
Critical Role ◽  
Cytosine Deaminase ◽  
Zinc Ion ◽  
Tumor Evolution ◽  
Active Site Residues

AbstractThe single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

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