scholarly journals Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design

2006 ◽  
Vol 399 (2) ◽  
pp. 199-204 ◽  
Author(s):  
Kamel EL Omari ◽  
Annelies Bronckaers ◽  
Sandra Liekens ◽  
Maria-Jésus Pérez-Pérez ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Thymidine Phosphorylase ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Structural Basis ◽  
Closed Conformation ◽  
Ribose Phosphate ◽  
Nucleoside Drugs ◽  
Competitive Product

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end com-plex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406–415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

scholarly journals Structural basis for glucose tolerance in GH1 β-glucosidases

2014 ◽  
Vol 70 (6) ◽  
pp. 1631-1639 ◽  
Author(s):  
Priscila Oliveira de Giuseppe ◽  
Tatiana de Arruda Campos Brasil Souza ◽  
Flavio Henrique Moreira Souza ◽  
Leticia Maria Zanphorlin ◽  
Carla Botelho Machado ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Active Site ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Product Inhibition ◽  
Structural Basis ◽  
Clear Correlation ◽  
Biomass Saccharification ◽  
Glucose Tolerant ◽  
Shed Light

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the −1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

scholarly journals Aldosterone Synthase Structure With Cushing Disease Drug LCI699 Highlights Avenues for Selective CYP11B Drug Design

Hypertension ◽  
2021 ◽  
Author(s):  
Simone Brixius-Anderko ◽  
Emily E. Scott
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Primary Aldosteronism ◽  
Therapeutic Option ◽  
Secondary Hypertension ◽  
Heme Iron ◽  
Structural Basis ◽  
Cushing Disease ◽  
Aldosterone Synthase ◽  
X Ray Crystallography

Primary aldosteronism, the major form of secondary hypertension, develops due to excess steroid hormone aldosterone produced by aldosterone synthase, also known as cytochrome P450 11B2. CYP11B2 is 93% identical to cortisol-producing CYP11B1, which makes it difficult to design selective drugs. LCI699 (Osilodrostat, Isturisa) was initially developed as a CYP11B2 inhibitor but due to poor selectivity was recently repurposed as the first Food and Drug Administration-approved drug for CYP11B1-mediated Cushing disease. Thus, there is still no effective therapeutic option targeting CYP11B2 for primary aldosteronism. Using a structure/function approach, aspects of LCI699 interaction with CYP11B2 were examined to facilitate the design of more selective inhibitors. LCI699 effectively binds and inhibits CYP11B2. To determine the structural basis for this interaction, X-ray crystallography was used to solve the structure of CYP11B2 bound to LCI699. LCI699 binds in the active site with its imidazole nitrogen coordinating the heme iron. LCI699 binding was compared with that of its analog fadrozole to both CYP11B enzymes. Comparison with the CYP11B1 structure reveals distinct CYP11B active site architectures which can be exploited to directed drug design. Exploiting structural differences between the CYP11B enzymes will promote the design of therapeutics for the treatment of primary aldosteronism targeting CYP11B2 while reducing undesirable side effects due to off-target CYP11B1 inhibition.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

scholarly journals Toward the Identification of Potential α-Ketoamide Covalent Inhibitors for SARS-CoV-2 Main Protease: Fragment-Based Drug Design and MM-PBSA Calculations

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 1004
Author(s):  
Mahmoud A. El Hassab ◽  
Mohamed Fares ◽  
Mohammed K. Abdel-Hamid Amin ◽  
Sara T. Al-Rashood ◽  
Amal Alharbi ◽  
...  
Keyword(s):  
Drug Design ◽  
Active Site ◽  
Binding Free Energy ◽  
Stable Complex ◽  
Active Site Residues ◽  
Structure Based Drug Design ◽  
Enzyme Active Site ◽  
Potential Inhibitor ◽  
Main Protease ◽  
Covalent Docking

Since December 2019, the world has been facing the outbreak of the SARS-CoV-2 pandemic that has infected more than 149 million and killed 3.1 million people by 27 April 2021, according to WHO statistics. Safety measures and precautions taken by many countries seem insufficient, especially with no specific approved drugs against the virus. This has created an urgent need to fast track the development of new medication against the virus in order to alleviate the problem and meet public expectations. The SARS-CoV-2 3CL main protease (Mpro) is one of the most attractive targets in the virus life cycle, which is responsible for the processing of the viral polyprotein and is a key for the ribosomal translation of the SARS-CoV-2 genome. In this work, we targeted this enzyme through a structure-based drug design (SBDD) protocol, which aimed at the design of a new potential inhibitor for Mpro. The protocol involves three major steps: fragment-based drug design (FBDD), covalent docking and molecular dynamics (MD) simulation with the calculation of the designed molecule binding free energy at a high level of theory. The FBDD step identified five molecular fragments, which were linked via a suitable carbon linker, to construct our designed compound RMH148. The mode of binding and initial interactions between RMH148 and the enzyme active site was established in the second step of our protocol via covalent docking. The final step involved the use of MD simulations to test for the stability of the docked RMH148 into the Mpro active site and included precise calculations for potential interactions with active site residues and binding free energies. The results introduced RMH148 as a potential inhibitor for the SARS-CoV-2 Mpro enzyme, which was able to achieve various interactions with the enzyme and forms a highly stable complex at the active site even better than the co-crystalized reference.

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

Kinetic studies of thymidine phosphorylase from mouse liver

Biochemistry ◽  
1985 ◽  
Vol 24 (24) ◽  
pp. 6799-6807 ◽  
Author(s):  
Max H. Iltzsch ◽  
Mahmoud H. El Kouni ◽  
Sungman Cha
Keyword(s):  
Mouse Liver ◽  
Thymidine Phosphorylase ◽  
Kinetic Studies

scholarly journals Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

2009 ◽  
Vol 284 (38) ◽  
pp. 25697-25703 ◽  
Author(s):  
Iain D. Kerr ◽  
Ji H. Lee ◽  
Christopher J. Farady ◽  
Rachael Marion ◽  
Mathias Rickert ◽  
...  
Keyword(s):  
Drug Design ◽  
Structural Basis ◽  
Antiparasitic Agents

scholarly journals Emergence of protocellular growth laws

2007 ◽  
Vol 362 (1486) ◽  
pp. 1841-1845 ◽  
Author(s):  
Tristan Rocheleau ◽  
Steen Rasmussen ◽  
Peter E Nielsen ◽  
Martin N Jacobi ◽  
Hans Ziock
Keyword(s):  
Kinetic Studies ◽  
Product Inhibition ◽  
Replication Process ◽  
Gene Replication ◽  
Math Biol ◽  
Curr Opin Cell Biol ◽  
Growth Laws ◽  
Internal Gene ◽  
The Many ◽  
The Individual

Template-directed replication is known to obey a parabolic growth law due to product inhibition (Sievers & Von Kiedrowski 1994 Nature 369 , 221; Lee et al . 1996 Nature 382 , 525; Varga & Szathmáry 1997 Bull. Math. Biol . 59 , 1145). We investigate a template-directed replication with a coupled template catalysed lipid aggregate production as a model of a minimal protocell and show analytically that the autocatalytic template–container feedback ensures balanced exponential replication kinetics; both the genes and the container grow exponentially with the same exponent. The parabolic gene replication does not limit the protocellular growth, and a detailed stoichiometric control of the individual protocell components is not necessary to ensure a balanced gene–container growth as conjectured by various authors (Gánti 2004 Chemoton theory ). Our analysis also suggests that the exponential growth of most modern biological systems emerges from the inherent spatial quality of the container replication process as we show analytically how the internal gene and metabolic kinetics determine the cell population's generation time and not the growth law (Burdett & Kirkwood 1983 J. Theor. Biol . 103 , 11–20; Novak et al . 1998 Biophys. Chem . 72 , 185–200; Tyson et al . 2003 Curr. Opin. Cell Biol . 15 , 221–231). Previous extensive replication reaction kinetic studies have mainly focused on template replication and have not included a coupling to metabolic container dynamics (Stadler et al . 2000 Bull. Math. Biol . 62 , 1061–1086; Stadler & Stadler 2003 Adv. Comp. Syst . 6 , 47). The reported results extend these investigations. Finally, the coordinated exponential gene–container growth law stemming from catalysis is an encouraging circumstance for the many experimental groups currently engaged in assembling self-replicating minimal artificial cells (Szostak 2001 et al . Nature 409 , 387–390; Pohorille & Deamer 2002 Trends Biotech . 20 123–128; Rasmussen et al . 2004 Science 303 , 963–965; Szathmáry 2005 Nature 433 , 469–470; Luisi et al . 2006 Naturwissenschaften 93 , 1–13). 1

Sign in / Sign up

Export Citation Format

Share Document