The unit was disbanded in 2018. It is part of the unit Laboratory of proteolytic enzyme chemistry.

Group of combinatorial methods for constructing biocatalysts

NamePositionContacts

Selected publications

  1. Terekhov S.S., Smirnov I.V., Stepanova A.V., Bobik T.V., Mokrushina Y.A., Ponomarenko N.A., Belogurov A.A. Jr, Rubtsova M.P., Kartseva O.V., Gomzikova M.O., Moskovtsev A.A., Bukatin A.S., Dubina M.V., Kostryukova E.S., Babenko V.V., Vakhitova M.T., Manolov A.I., Malakhova M.V., Kornienko M.A., Tyakht A.V., Vanyushkina A.A., Ilina E.N., Masson P., Gabibov A.G., Altman S. (2017). Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity. Proc. Natl. Acad. Sci. U.S.A. 114 (10), 2550–2555 [+]

    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.

    ID:1962
  2. Smirnov I.V., Golovin A.V., Chatziefthimiou S.D., Stepanova A.V., Peng Y., Zolotareva O.I., Belogurov A.A. Jr, Kurkova I.N., Ponomarenko N.A., Wilmanns M., Blackburn G.M., Gabibov A.G., Lerner R.A. (2016). Robotic QM/MM-driven maturation of antibody combining sites. Sci Adv 2 (10), e1501695 [+]

    In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

    ID:1605
  3. Belogurov A., Zakharov K., Lomakin Y., Surkov K., Avtushenko S., Kruglyakov P., Smirnov I., Makshakov G., Lockshin C., Gregoriadis G., Genkin D., Gabibov A., Evdoshenko E. (2016). CD206-Targeted Liposomal Myelin Basic Protein Peptides in Patients with Multiple Sclerosis Resistant to First-Line Disease-Modifying Therapies: A First-in-Human, Proof-of-Concept Dose-Escalation Study. Neurotherapeutics , [+]

    Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBP46-62, MBP124-139 and MBP147-170 (Xemys) suppressed experimental autoimmune encephalomyelitis in dark Agouti rats. The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe and well tolerated when administered for 6 weeks to a maximum single dose of 900 μg. Expanded Disability Status Scale scores and numbers of T2-weighted and new gadolinium-enhancing lesions on magnetic resonance imaging were statistically unchanged at study exit compared with baseline; nonetheless, the increase of number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was statistically significant. During treatment, the serum concentrations of the cytokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-7 decreased, whereas the level of tumor necrosis factor-α increased. These results provide evidence for the further development of Xemys as an antigen-specific, disease-modifying therapy for patients with MS.

    ID:1535

Ivan Smirnov

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 31, office. 607
  • Phone: +7(926)7397865
  • E-mail: ivansmr@inbox.ru

Development of ultrahigh-throughput method for screening of catalytic activity. Validation. (2017-11-28)

The research is aimed at the search and creation of catalytic biological antidotes. To find new catalytic antidotes based on butyrylcholinesterase, a panel of libraries of the active center of BuChE was created. The library of 284-288 loop was successfully used to validate the method of ultrahigh-throughput screening of biocatalytic activity in droplets of a microfluidic emulsion. The obtained results, together with the developed quantum-mechanical algorithm for calculating the dephosphorylation step of covalent BChE-paraoxon complex, made it possible to create an optimized library for the BChE RKH-4 yeast display, representing 1 * 10 ^ 6 variants.

Publications

  1. Terekhov S.S., Smirnov I.V., Stepanova A.V., Bobik T.V., Mokrushina Y.A., Ponomarenko N.A., Belogurov A.A. Jr, Rubtsova M.P., Kartseva O.V., Gomzikova M.O., Moskovtsev A.A., Bukatin A.S., Dubina M.V., Kostryukova E.S., Babenko V.V., Vakhitova M.T., Manolov A.I., Malakhova M.V., Kornienko M.A., Tyakht A.V., Vanyushkina A.A., Ilina E.N., Masson P., Gabibov A.G., Altman S. (2017). Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity. Proc. Natl. Acad. Sci. U.S.A. 114 (10), 2550–2555 [+]

    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.

    ID:1962

Generation of enantioselective bioscavengers based on antibodies. (2017-11-28)

In the framework of the study of catalytic antidotes based on antibodies, an algorithm for predicting mutants capable of selectively interacting with (R) and (S) isomers of PCT was proposed.

Publications

  1. Golovin A.V., Smirnov I.V., Stepanova A.V., Zalevskiy A.O., Zlobin A.S., Ponomarenko N.A., Belogurov A.A. Jr, Knorre V.D., Hurs E.N., Chatziefthimiou S.D., Wilmanns M., Blackburn G.M., Khomutov R.M., Gabibov A.G. (2017). Evolution of catalytic centers of antibodies by virtual screening of broad repertoire of mutants using supercomputer. Dokl. Biochem. Biophys. 475 (1), 245–249 [+]

    It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.

    ID:1983