Belogurov AA, Jr.

References (7)

Title : Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers - Mokrushina_2020_Proc.Natl.Acad.Sci.U.S.A_117_22841
Author(s) : Mokrushina YA , Golovin AV , Smirnov IV , Chatziefthimiou SD , Stepanova AV , Bobik TV , Zalevsky AO , Zlobin AS , Konovalov KA , Terekhov SS , Stepanov AV , Pipiya SO , Shamborant OG , Round E , Belogurov AA, Jr. , Bourenkov G , Makarov AA , Wilmanns M , Xie J , Blackburn GM , Gabibov AG , Lerner RA
Ref : Proc Natl Acad Sci U S A , 117 :22841 , 2020
Abstract : Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.
ESTHER : Mokrushina_2020_Proc.Natl.Acad.Sci.U.S.A_117_22841
PubMedSearch : Mokrushina_2020_Proc.Natl.Acad.Sci.U.S.A_117_22841
PubMedID: 32859757

Title : Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity - Terekhov_2017_Proc.Natl.Acad.Sci.U.S.A_114_2550
Author(s) : Terekhov SS , Smirnov IV , Stepanova AV , Bobik TV , Mokrushina YA , Ponomarenko NA , Belogurov AA, Jr. , Rubtsova MP , Kartseva OV , Gomzikova MO , Moskovtsev AA , Bukatin AS , Dubina MV , Kostryukova ES , Babenko VV , Vakhitova MT , Manolov AI , Malakhova MV , Kornienko MA , Tyakht AV , Vanyushkina AA , Ilina EN , Masson P , Gabibov AG , Altman S
Ref : Proc Natl Acad Sci U S A , 114 :2550 , 2017
Abstract : Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
ESTHER : Terekhov_2017_Proc.Natl.Acad.Sci.U.S.A_114_2550
PubMedSearch : Terekhov_2017_Proc.Natl.Acad.Sci.U.S.A_114_2550
PubMedID: 28202731

Title : A novel expression cassette delivers efficient production of exclusively tetrameric human butyrylcholinesterase with improved pharmacokinetics for protection against organophosphate poisoning - Terekhov_2015_Biochimie_118_51
Author(s) : Terekhov SS , Smirnov IV , Bobik TV , Shamborant OG , Zenkova MA , Chernolovskaya EL , Gladkikh DV , Murashev AN , Dyachenko IA , Palikov VA , Palikova YA , Knorre VD , Belogurov AA, Jr. , Ponomarenko NA , Blackburn GM , Masson P , Gabibov AG
Ref : Biochimie , 118 :51 , 2015
Abstract : Butyrylcholinesterase is a stoichiometric bioscavenger against poisoning by organophosphorus pesticides and nerve agents. The low level of expression and extremely rapid clearance of monomeric recombinant human butyrylcholinesterase (rhBChE) from bloodstream (t(1/2) approximately 2 min) limits its pharmaceutical application. Recently (Ilyushin at al., PNAS, 2013) we described a long-acting polysialylated recombinant butyrylcholinesterase (rhBChE-CAO), stable in the bloodstream, that protects mice against 4.2 LD50 of VR. Here we report a set of modifications of the initial rhBChE expression vector to improve stability of the enzyme in the bloodstream and increase its production in CHO cells by introducing in the expression cassette: (i) the sequence of the natural human PRAD-peptide in frame with rhBChE gene via "self-processing" viral F2A peptide under control of an hEF/HTLV promoter, and (ii) previously predicted in silico MAR 1-68 and MAR X-29 sequences. This provides fully tetrameric rhBChE (4rhBChE) at 70 mg/l, that displays improved pharmacokinetics (t(1/2) = 32 +/- 1.2 h, MRT = 43 +/- 2 h). 3D Fluorescent visualization and distribution of (125)I-labeled enzyme reveals similar low level 4rhBChE and rhBChE-CAO accumulation in muscle, fat, and brain. Administered 4rhBChE was mainly catabolized in the liver and breakdown products were excreted in kidney. Injection of 1.2 LD50 and 1.1 LD50 of paraoxon to BALB/c and knockout BChE-/- mice pre-treated with 4rhBChE (50 mg/kg) resulted in 100% and 78% survival, respectively, without perturbation of long-term behavior. In contrast, 100% mortality of non-pre-treated mice was observed. The high expression level of 4rhBChE in CHO cells permits consideration of this new expression system for manufacturing BChE as a biopharmaceutical.
ESTHER : Terekhov_2015_Biochimie_118_51
PubMedSearch : Terekhov_2015_Biochimie_118_51
PubMedID: 26239905

Title : Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo - Ilyushin_2013_Proc.Natl.Acad.Sci.U.S.A_110_1243
Author(s) : Ilyushin DG , Smirnov IV , Belogurov AA, Jr. , Dyachenko IA , Zharmukhamedova T , Novozhilova TI , Bychikhin EA , Serebryakova MV , Kharybin ON , Murashev AN , Anikienko KA , Nikolaev EN , Ponomarenko NA , Genkin DD , Blackburn GM , Masson P , Gabibov AG
Ref : Proc Natl Acad Sci U S A , 110 :1243 , 2013
Abstract : The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.
ESTHER : Ilyushin_2013_Proc.Natl.Acad.Sci.U.S.A_110_1243
PubMedSearch : Ilyushin_2013_Proc.Natl.Acad.Sci.U.S.A_110_1243
PubMedID: 23297221

Title : Strategies for the selection of catalytic antibodies against organophosphorus nerve agents - Smirnov_2013_Chem.Biol.Interact_203_196
Author(s) : Smirnov IV , Belogurov AA, Jr. , Friboulet A , Masson P , Gabibov AG , Renard PY
Ref : Chemico-Biological Interactions , 203 :196 , 2013
Abstract : Among the strategies aimed at biocompatible means for organophosphorus nerve agents neutralization, immunoglobulins have attracted attention in the 1990's and 2000's both for their ability to immobilize the toxicants, but also for their ability to be turned into enzymatically active antibodies known as catalytic antibodies or abzymes (antibodies - enzymes). We will present here a critical review of the successive strategies used for the selection of these nerve agent-hydrolyzing abzymes, based on hapten design, namely antibodies raised against a wide variety of transition state analogs, and eventually the strategies based on anti-idiotypic antibodies and reactibodies.
ESTHER : Smirnov_2013_Chem.Biol.Interact_203_196
PubMedSearch : Smirnov_2013_Chem.Biol.Interact_203_196
PubMedID: 23123255

Title : Creation of catalytic antibodies metabolizing organophosphate compounds - Kurkova_2012_Biochemistry.(Mosc)_77_1139
Author(s) : Kurkova IN , Smirnov IV , Belogurov AA, Jr. , Ponomarenko NA , Gabibov AG
Ref : Biochemistry (Mosc) , 77 :1139 , 2012
Abstract : Development of new ways of creating catalytic antibodies possessing defined substrate specificity towards artificial substrates has important fundamental and practical aspects. Low immunogenicity combined with high stability of immunoglobulins in the blood stream makes abzymes potent remedies. A good example is the cocaine-hydrolyzing antibody that has successfully passed clinical trials. Creation of an effective antidote against organophosphate compounds, which are very toxic substances, is a very realistic goal. The most promising antidotes are based on cholinesterases. These antidotes are now expensive, and their production methods are inefficient. Recombinant antibodies are widely applied in clinics and have some advantage compared to enzymatic drugs. A new potential abzyme antidote will combine effective catalysis comparable to enzymes with high stability and the ability to switch on effector mechanisms specific for antibodies. Examples of abzymes metabolizing organophosphate substrates are discussed in this review.
ESTHER : Kurkova_2012_Biochemistry.(Mosc)_77_1139
PubMedSearch : Kurkova_2012_Biochemistry.(Mosc)_77_1139
PubMedID: 23157294

Title : Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics - Smirnov_2011_Proc.Natl.Acad.Sci.U.S.A_108_15954
Author(s) : Smirnov IV , Carletti E , Kurkova I , Nachon F , Nicolet Y , Mitkevich VA , Debat H , Avalle B , Belogurov AA, Jr. , Kuznetsov N , Reshetnyak A , Masson P , Tonevitsky AG , Ponomarenko NA , Makarov AA , Friboulet A , Tramontano A , Gabibov AG
Ref : Proc Natl Acad Sci U S A , 108 :15954 , 2011
Abstract : Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-A-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.
ESTHER : Smirnov_2011_Proc.Natl.Acad.Sci.U.S.A_108_15954
PubMedSearch : Smirnov_2011_Proc.Natl.Acad.Sci.U.S.A_108_15954
PubMedID: 21896761