Смирнов Иван Витальевич

Доктор химических наук

Старший научный сотрудник (Лаборатория биокатализа), Руководитель подразделения (Группа комбинаторных методов конструирования биокатализаторов)

Тел.: +7 (926) 7397865

Эл. почта: ivansmr@inbox.ru

Избранные публикации

  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 [+]

    Каталитические антитела (абзимы) – представляют уникальную возможность осуществлять широкий спектр химических превращений благодаря способности структурной гиперизменчивости молекул иммуноглобулинов (Ig) [1,2]. С помощью абзимов удалось осуществить катализ реакций, не имеющих соответствующих ферментативных биокаталитических партнеров, в частности ряд превращений фосфорорганических отравляющих веществ (ФОТ) [3,4]. Вместе с тем катализ антителами имеет ряд существенных недостатков, связанных с попытками получения «иммунологических импринтов» аналогов переходных состояний химических превращений и, как следствие, реализацию весьма скромных параметров каталитического превращения по сравнению с некаталитическим процессом. В этой связи нами предложено осуществлять квантово-механические/молекулярно-динамические расчеты химических реакций, которые предполагается катализировать антителами, а затем осуществлять виртуальный скрининг библиотеки потенциальных мутантов антител с задачей выбора оптимального биокатализатора. Эффективность данного подхода проверена нами на примере гидролиза ФОТ параоксона как с помощью кинетических подходов, так и с применением рентгеноструктурного анализа, созданного de novo антительного биокатализатора.

  2. 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.

  3. 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.

  4. 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.

  5. Ponomarenko N., Chatziefthimiou S.D., Kurkova I., Mokrushina Y., Stepanova A., Smirnov I., Avakyan M., Bobik T., Mamedov A., Mitkevich V., Belogurov A., Fedorova O.S., Dubina M., Golovin A., Lamzin V., Friboulet A., Makarov A.A., Wilmanns M., Gabibov A. (2014). Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody. Acta Crystallogr. D Biol. Crystallogr. 70 (Pt 3), 708–19 [+]

    The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.

  6. Lomakin Y.A., Zakharova M.Y., Stepanov A.V., Dronina M.A., Smirnov I.V., Bobik T.V., Pyrkov A.Y., Tikunova N.V., Sharanova S.N., Boitsov V.M., Vyazmin S.Y., Kabilov M.R., Tupikin A.E., Krasnov A.N., Bykova N.A., Medvedeva Y.A., Fridman M.V., Favorov A.V., Ponomarenko N.A., Dubina M.V., Boyko A.N., Vlassov V.V., Belogurov A.A. Jr, Gabibov A.G. (2014). Heavy-light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation. Mol. Immunol. , [+]

    The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

  7. Kuzina E., Kudriaeva A., Smirnov I., Dubina M.V., Gabibov A., Belogurov A. (2014). Glatiramer Acetate and Nanny Proteins Restrict Access of the Multiple Sclerosis Autoantigen Myelin Basic Protein to the 26S Proteasome. Biomed Res Int 2014, 926394 [+]

    We recently showed that myelin basic protein (MBP) is hydrolyzed by 26S proteasome without ubiquitination. The previously suggested concept of charge-mediated interaction between MBP and the proteasome led us to attempt to compensate or mimic its positive charge to inhibit proteasomal degradation. We demonstrated that negatively charged actin and calmodulin (CaM), as well as basic histone H1.3, inhibit MBP hydrolysis by competing with the proteasome and MBP, respectively, for binding their counterpart. Interestingly, glatiramer acetate (GA), which is used to treat multiple sclerosis (MS) and is structurally similar to MBP, inhibits intracellular and in vitro proteasome-mediated MBP degradation. Therefore, the data reported in this study may be important for myelin biogenesis in both the normal state and pathophysiological conditions.

  8. Ilyushin D.G., Smirnov I.V., Belogurov A.A. Jr, Dyachenko I.A., Zharmukhamedova T.I.u., Novozhilova T.I., Bychikhin E.A., Serebryakova M.V., Kharybin O.N., Murashev A.N., Anikienko K.A., Nikolaev E.N., Ponomarenko N.A., Genkin D.D., Blackburn G.M., Masson P., Gabibov A.G. (2013). Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo. Proc. Natl. Acad. Sci. U.S.A. 110 (4), 1243–8 [+]

    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.

  9. Belogurov A., Smirnov I., Ponomarenko N., Gabibov A. (2012). Antibody-antigen pair probed by combinatorial approach and rational design: bringing together structural insights, directed evolution, and novel functionality. FEBS Lett. 586 (18), 2966–73 [+]

    The unique hypervariability of the immunoglobulin (Ig) superfamily provides a means to create both binding and catalytic antibodies with almost any desired specificity and activity. The diversity of antigens and concept of adaptive response suggest that it is possible to find an antigen pair to any raised Ig. In the current review we discuss combinatorial approaches, which makes it possible to obtain an antibody with predefined properties, followed by 3D structure-based rational design to enhance or dramatically change its characteristics. A similar strategy, but applied to the second partner of the antibody-antigen pair, may result in selection of complementary substrates to the chosen Ig. Finally, 2D screening may be performed solving the "Chicken and Egg" problem when neither antibody nor antigen is known.