Design,structure-based focusing and in silico screening of combinatorial library of peptidomimetic inhibitors of Dengue virus NS2B-NS3 protease

Abstract  

Serine protease activity of the NS3 protein of Dengue virus is an important target of antiviral agents that interfere with the viral polyprotein precursor processing catalyzed by the NS3 protease (NS3pro), which is important for the viral replication and maturation. Recent studies showed that substrate-based peptidomimetics carrying an electrophilic warhead inhibit the NS2B-NS3pro cofactor-protease complex with inhibition constants in the low micromolar concentration range when basic amino acid residues occupy P₁ and P₂ positions of the inhibitor, and an aldehyde warhead is attached to the P₁. We have used computer-assisted combinatorial techniques to design, focus using the NS2B-NS3pro receptor 3D structure, and in silico screen a virtual library of more than 9,200 peptidomimetic analogs targeted around the template inhibitor Bz-Nle-Lys-Arg-Arg-H (Bz—benzoyl) that are composed mainly of unusual amino acid residues in all positions P₁–P₄. The most promising virtual hits were analyzed in terms of computed enzyme-inhibitor interactions and Adsorption, Distribution, Metabolism and Excretion (ADME) related physico-chemical properties. Our study can direct the interest of medicinal chemists working on a next generation of antiviral chemotherapeutics against the Dengue Fever towards the explored subset of the chemical space that is predicted to contain peptide aldehydes with NS3pro inhibition potencies in nanomolar range which display ADME-related properties comparable to the training set inhibitors.     

Structure-guided fragment-based<Emphasis Type="Italic"> in silico</Emphasis> drug design of dengue protease inhibitors

An in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC₅₀ = 7.7 μM and 37.9 μM, respectively) and the related West Nile virus protease (IC₅₀ = 6.3 μM and 39.0 μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters.     

NMR Analysis of Apo Glutamine‐Binding Protein Exposes Challenges in the Study of Interdomain Dynamics

Glutamine‐binding protein (GlnBP) displays an apo, “open” and a holo, “closed” crystal form, mutually related by a rigid‐body reorientation of its domains. A fundamental question about such large‐scale conformational transitions, whether the closed state exists in the absence of ligand, is controversial in the case of GlnBP. NMR observations have indicated no evidence of the closed form, whereas experimentally validated computations have suggested a remarkable ca. 40 % population. Herein, a paramagnetic NMR strategy designed to detect the putative apo‐closed species shows that a major population of the latter is highly improbable. Further, NMR residual dipolar couplings collected under three anisotropic conditions do not reveal differential domain alignment and establish that the average solution conformation is satisfied by the apo‐open crystal structure. Our results indicate that the computational prediction of large‐scale interdomain motions is not trivial and may lead to erroneous conclusions without proper experimental validation.     

Conformationally Flexible Bis(9‐fluorenylidene)porphyrin Diradicaloids

A stable 5,10‐bis(9‐fluorenylidene)porphyrin (Por‐Fl) diradicaloid was synthesized. It shows a quinoidal, saddle‐shaped geometry in the single crystal but can be thermally populated to a triplet diradical both in solution and in the solid state. Coordination with the Ni²⁺ ion (Por‐Fl‐Ni) does not significantly change the contorted conformation but reduces the singlet–triplet gap. Heat‐induced geometric change can explain the observed paramagnetic properties as well as unusual hysteresis in SQUID measurements. On the other hand, protonation (Por‐Fl‐2H⁺) dramatically changes the conformation while maintains the closed‐shell electronic structure. Our studies demonstrate how heat, coordination, and protonation affect the geometry, diradical character, and physical properties of conformationally flexible open‐shell singlet diradicaloids.     

Hepatitis C virus NS3 protease requires its NS4A cofactor peptide for optimal binding of a boronic acid inhibitor as shown by NMR

NMR spectroscopy was used to characterize the hepatitis C virus (HCV) NS3 protease in a complex with the 24 residue peptide cofactor from NS4A and a boronic acid inhibitor, Ac-Asp-Glu-Val-Val-Pro-boroAlg-OH. Secondary-structure information, NOE constraints between protease and cofactor, and hydrogen-deuterium exchange rates revealed that the cofactor was an integral strand in the N-terminal beta-sheet of the complex as observed in X-ray crystal structures. Based upon chemical-shift perturbations, inhibitor-protein NOEs, and the protonation state of the catalytic histidine, the boronic acid inhibitor was bound in the substrate binding site as a transition state mimic. In the absence of cofactor, the inhibitor had a lower affinity for the protease. Although the inhibitor binds in the same location, differences were observed at the catalytic site of the protease.     

Doxycycline Interferes with Zika Virus Serine Protease and Inhibits Virus Replication in Human Skin Fibroblasts

Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (−7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.     

Design, synthesis, and in vitro evaluation of potential West Nile virus protease inhibitors based on the 1-oxo-1,2,3,4-tetrahydroisoquinoline and 1-oxo-1,2-dihydroisoquinoline scaffolds

The 1-oxo-1, 2, 3, 4-tetrahydroisoquinoline and 1-Oxo-1, 2-dihydroisoquinoline scaffolds were utilized in the design and solution phase synthesis of focused libraries of compounds for screening against West Nile Virus (WNV) protease. Exploratory studies have led to the identification of a WNV protease inhibitor (a 1-oxo-1, 2-dihydroisoquinoline-based derivative, 12j) which could potentially serve as a launching pad for a hit-to-lead optimization campaign. The identified hit was devoid of any inhibitory activity toward a panel of mammalian serine proteases.     

The open structure of a multi-drug-resistant HIV-1 protease is stabilized by crystal packing contacts

The introduction of HIV-1 protease (HIV-PR) inhibitors has led to a dramatic increase in patient survival; however, these gains are threatened by the emergence of multi-drug-resistant strains. Design of inhibitors that overcome resistance would be greatly facilitated by deeper insight into the mechanistic events associated with binding of substrates and inhibitors, as well as an understanding of the effects of resistance mutations on the structure and dynamic behavior of HIV-PR. We previously reported a series of simulations that provide a model for HIV-PR dynamics, with spontaneous conversions between the bound and unbound crystal forms upon addition or removal of an inhibitor. Importantly, the unbound protease transiently sampled a third fully open state that permits entry to the active site, unlike both crystallographic forms. Recently, a crystal structure of unbound HIV-PR was reported for the MDR 769 isolate (PDB: 1TW7); unlike all previous experimental structures, the binding pocket is open. It is suggested that drug resistance in this strain arises at least in part from the inability of inhibitors to induce closing. We carried out simulations of the MDR 769 HIV-PR mutant and observed that the reported structure is unstable in solution and rapidly adopts the semi-open conformation observed for the unbound wild-type protease in solution. Further analysis suggests that the wide-open structure observed for MDR 769 arises not from sequence variation, but instead is an artifact from crystal packing. Thus, despite being the first experimental structure to reveal flap opening sufficient for substrate access to the active site, this structure may not be directly relevant to studies of inhibitor entry or to the cause of HIV-PR drug resistance.     

Rapid measurement of pseudocontact shifts in metalloproteins by proton-detected solid-state NMR spectroscopy

Pseudocontact shifts (PCSs) arise in paramagnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of (1)H-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved (1)H PCSs, in a large metalloprotein, Co(2+)-substituted superoxide dismutase (Co(2+)-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with (1)H-(1)H distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.     

Synthesis and resolution of diethyl (1S,2S)-1-amino-2-vinylcyclopropane-1-phosphonate for HCV NS3 protease inhibitors

We herein describe an efficient synthesis of optically active diethyl 1-amino-2-vinylcyclopropane-1-phosphonate (analogous to 1-amino-2-vinylcyclopropane-1-carboxylate). The racemic phosphonate diethyl ester was obtained from an imine derived from aminomethylphosphonate diester and trans-1,4-dibromo-2-butene. Crystallizations of the dibenzoyl-l-tartaric acid salt allowed for separation of enantiomers. The enantiomerically pure material was used to synthesize an extremely potent tripeptide phosphonate inhibitor of HCV NS3 protease. X-ray crystal structure of the inhibitor bound to the HCV NS3 protease confirmed the absolute stereochemistry of the title compound.     

HIV-1 protease flaps spontaneously close to the correct structure in simulations following manual placement of an inhibitor into the open state

We report unrestrained, all-atom molecular dynamics simulations of HIV-1 protease (HIV-PR) with a continuum solvent model that reproducibly sample closing of the active site flaps following manual placement of a cyclic urea inhibitor into the substrate binding site of the open protease. The open form was obtained from the unbound, semi-open HIV-PR crystal structure, which we recently reported (Hornak, V.; et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 915-920.) to have spontaneously opened during unrestrained dynamics. In those simulations, the transiently open flaps always returned to the semi-open form that is observed in all crystal structures of the free protease. Here, we show that manual docking of the inhibitor reproducibly induces spontaneous conversion to the closed form as seen in all inhibitor-bound HIV-PR crystal structures. These simulations reproduced not only the greater degree of flap closure, but also the striking difference in flap "handedness" between bound and free enzyme. In most of the simulations, the final structures were highly accurate. Root-mean-square deviations (RMSD) from the crystal structure of the complex were approximately 1.5 A (averaged over the last 100 ps) for the inhibitor and each flap despite initial RMSD of 2-5 A for the inhibitors and 6-11 A for the flaps. Key hydrogen bonds were formed between the flap tips and between flaps and inhibitor that match those seen in the crystal structure. The results demonstrate that all-atom simulations have the ability to significantly improve poorly docked ligand conformations and reproduce large-scale receptor conformational changes that occur upon binding.     

Pseudo‐Contact NMR Shifts over the Paramagnetic Metalloprotein CoMMP‐12 from First Principles

Long‐range pseudo‐contact NMR shifts (PCSs) provide important restraints for the structure refinement of proteins when a paramagnetic metal center is present, either naturally or introduced artificially. Here we show that ab initio quantum‐chemical methods and a modern version of the Kurland–McGarvey approach for paramagnetic NMR (pNMR) shifts in the presence of zero‐field splitting (ZFS) together provide accurate predictions of all PCSs in a metalloprotein (high‐spin cobalt‐substituted MMP‐12 as a test case). Computations of 314 ¹³C PCSs using g‐ and ZFS tensors based on multi‐reference methods provide a reliable bridge between EPR‐parameter‐ and susceptibility‐based pNMR formalisms. Due to the high sensitivity of PCSs to even small structural differences, local structures based either on X‐ray diffraction or on various DFT optimizations could be evaluated critically by comparing computed and experimental PCSs. Many DFT functionals provide insufficiently accurate structures. We also found the available 1RMZ PDB X‐ray structure to exhibit deficiencies related to binding of a hydroxamate inhibitor. This has led to a newly refined PDB structure for MMP‐12 (5LAB) that provides a more accurate coordination arrangement and PCSs.     

Conformational study on telaprevir by HPLC–DAD–MS and theoretical calculation

Telaprevir is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3‐4A serine protease. it is used for the treatment of HCV infection in combination with peginterferon alfa and ribavirin. In the present work, the E–Z isomerization process of telaprevir in solution was revealed by online HPLC–DAD (diode array detector)–MS, variable‐temperature and variable‐gradient experiments. The molecular geometry information of the two isomers was established by molecular mechanics calculations, and good correlation between the two isomers' UV–vis spectra and their molecular geometry information was also discovered. In addition, it was revealed by molecular docking that the two isomers have different affinities to HCV NS3?4A protease, and the Z isomer, the minor form of telaprevir in solution, is the more effective inhibitor of HCV NS3?4A protease. The investigation can provide more structure information about telaprevir in solution and in the binding process of HCV NS3?4A protease.     

Design and synthesis of 6-amino-5-oxo-1,2,3,5-tetrahydro-3-indolizinecarboxylic acids as β-sheet peptidomimetics

6-Amino-5-oxo-1,2,3,5-tetrahydro-3-indolizinecarboxylic acid was designed as a novel scaffold that can effectively mimic the extended conformation of a peptide. The key reaction in the synthesis of the scaffold involved a [3+2]-cycloaddition of a dicarbonyl stabilized isomünchnone intermediate. Its effectiveness as a β-sheet mimetic was demonstrated by the preparation of a potent HCV NS3 protease inhibitor.     

Solid-state NMR crystallography through paramagnetic restraints

Pseudocontact shifts (PCSs) measured by solid-state NMR spectroscopy (SS-NMR) on microcrystalline powders of a paramagnetic metalloprotein permit NMR crystallography. Along with other restraints for SS-NMR experiments, the protein molecular structure as well as the correct crystal packing are obtained.     

Crystal structure and molecular conformation of E-64, a cysteine protease inhibitor

In order to elucidate the conformational characteristics of cysteine protease inhibitors contributing to their inhibitory activities, the conformation of E-64 (N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine), a potent inhibitor of papain, was determined by X-ray crystal structure analysis. The molecules were packed in the crystal through electrostatic forces and hydrogen bonding between the oppositely charged terminal groups and between the amide groups. Two crystallographically independent E-64 molecules both took a flattened and slightly curved structure, which is similar to that of loxistatin, a related cysteine protease inhibitor. Based on the present results, a possible inhibitory mechanism of E-64 is proposed, with reference to the binding mode observed in the crystal structure of papain-substrate analogue complex.     

Relative stability of the open and closed conformations of the active site loop of streptavidin

The eight-residue surface loop, 45-52 (Ser, Ala, Val, Gly, Asn, Ala, Glu, Ser), of the homotetrameric protein streptavidin has a "closed" conformation in the streptavidin-biotin complex, where the corresponding binding affinity is one of the strongest found in nature (ΔG ～ -18 kcal∕mol). However, in most of the crystal structures of apo (unbound) streptavidin, the loop conformation is "open" and typically exhibits partial disorder and high B-factors. Thus, it is plausible to assume that the loop structure is changed from open to closed upon binding of biotin, and the corresponding difference in free energy, ΔF = F(open) - F(closed) in the unbound protein, should therefore be considered in the total absolute free energy of binding. ΔF (which has generally been neglected) is calculated here using our "hypothetical scanning molecular-dynamics" (HSMD) method. We use a protein model in which only the atoms closest to the loop are considered (the "template") and they are fixed in the x-ray coordinates of the free protein; the x-ray conformation of the closed loop is attached to the same (unbound) template and both systems are capped with the same sphere of TIP3P water. Using the force field of the assisted model building with energy refinement (AMBER), we carry out two separate MD simulations (at temperature T = 300 K), starting from the open and closed conformations, where only the atoms of the loop and water are allowed to move (the template-water and template-loop interactions are considered). The absolute F(open) and F(closed) (of loop + water) are calculated from these trajectories, where the loop and water contributions are obtained by HSMD and a thermodynamic integration (TI) process, respectively. The combined HSMD-TI procedure leads to total (loop + water) ΔF = -27.1 ± 2.0 kcal∕mol, where the entropy TΔS constitutes 34% of ΔF, meaning that the effect of S is significant and should not be ignored. Also, ΔS is positive, in accord with the high flexibility of the open loop observed in crystal structures, while the energy ΔE is unexpectedly negative, thus also adding to the stability of the open loop. The loop and the 250 capped water molecules are the largest system studied thus far, which constitutes a test for the efficiency of HSMD-TI; this efficiency and technical issues related to the implementation of the method are also discussed. Finally, the result for ΔF is a prediction that will be considered in the calculation of the absolute free energy of binding of biotin to streptavidin, which constitutes our next project.     

External-stimuli responsive photophysics and liquid crystal properties of self-assembled "phosphole-lipids"

A series of new amphiphilic phosphonium materials that combine the electronic features of phospholes with self-assembly features of lipids were synthesized. Variable concentration/temperature and 2D NMR studies suggested that the systems undergo intramolecular conformation changes between a "closed" and "open" form that are triggered by intermolecular interactions. The amphiphilic features of the phospholium species also induce liquid crystalline and soft crystal phase behavior in the solid state, which was studied by differential scanning calorimetry (DSC), polarized optical microscopy (POM), and variable temperature powder X-ray diffraction (VT-PXRD). The studies revealed that both conjugated backbones and counteranions work together to organize the systems into different morphologies (liquid crystal/soft crystal). Dithieno[3,2-b:2',3'-d]phosphole-based compounds exhibit enhanced emission in the solid state and at low temperature in solution due to aggregation-induced enhanced emission (AIEE). Photoinduced electron transfer (PET) induced via the alkoxybenzyl group at the phosphonium center in the fused-ring systems can be effectively suppressed through intermolecular charge transfer (ICT) processes within the main scaffold of a nonfused system, which was confirmed by static and dynamic fluorescence spectroscopy. The dynamic features of these new materials also endow the systems with external-stimuli responsive photophysical properties that can be triggered by temperature and/or mechanical forces.     

Trinuclear gold(I) triazolates: a new class of wide-band phosphors and sensors

A new cyclic gold(I) triazolate trimer, [Au(3,5-i-Pr2Tz)]3 (1), exhibits fully overlapping aurophilically bonded dimer-of-trimer units that lead to multiple phosphorescence bands in both the solid state and solution. The conformation of the hexanuclear unit exhibits reversible interconversion between C2 and D3 effective symmetries, depending on the crystal temperature or solution concentration, the variation of which leads to isoemissive and isosbestic points. Solutions of 1 exhibit remarkable quenching properties that demonstrate molecular recognition with high selectivity and hypersensitivity for some reagents, as influenced by protonation via Br?nsted acids, pi intercalation, and/or energy transfer. The quenched phosphorescence of 1 by acetic acids can be regenerated by NEt3.     

The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.