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Dive into the research topics where Alexey Lugovskoy is active.

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Featured researches published by Alexey Lugovskoy.


Antimicrobial Agents and Chemotherapy | 2009

Identification and Characterization of Mefloquine Efficacy against JC Virus In Vitro

Margot Brickelmaier; Alexey Lugovskoy; Ramya Kartikeyan; Marta M. Reviriego-Mendoza; Norm Allaire; Kenneth J. Simon; Richard J. Frisque; Leonid Gorelik

ABSTRACT Progressive multifocal leukoencephalopathy (PML) is a rare but frequently fatal disease caused by the uncontrolled replication of JC virus (JCV), a polyomavirus, in the brains of some immunocompromised individuals. Currently, no effective antiviral treatment for this disease has been identified. As a first step in the identification of such therapy, we screened the Spectrum collection of 2,000 approved drugs and biologically active molecules for their anti-JCV activities in an in vitro infection assay. We identified a number of different drugs and compounds that had significant anti-JCV activities at micromolar concentrations and lacked cellular toxicity. Of the compounds with anti-JCV activities, only mefloquine, an antimalarial agent, has been reported to show sufficiently high penetration into the central nervous system such that it would be predicted to achieve efficacious concentrations in the brain. Additional in vitro experiments demonstrated that mefloquine inhibits the viral infection rates of three different JCV isolates, JCV(Mad1), JCV(Mad4), and JCV(M1/SVEΔ), and does so in three different cell types, transformed human glial (SVG-A) cells, primary human fetal glial cells, and primary human astrocytes. Using quantitative PCR to quantify the number of viral copies in cultured cells, we have also shown that mefloquine inhibits viral DNA replication. Finally, we demonstrated that mefloquine does not block viral cell entry; rather, it inhibits viral replication in cells after viral entry. Although no suitable animal model of PML or JCV infection is available for the testing of mefloquine in vivo, our in vitro results, combined with biodistribution data published in the literature, suggest that mefloquine could be an effective therapy for PML.


Protein Science | 2006

Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design

Louis A. Clark; P. Ann Boriack-Sjodin; John K. Eldredge; Christopher Fitch; Bethany Friedman; Karl Hanf; Matthew Jarpe; Stefano F. Liparoto; You Li; Alexey Lugovskoy; Stephan Miller; Mia Rushe; Woody Sherman; Kenneth J. Simon; Herman W. T. van Vlijmen

Improving the affinity of a high‐affinity protein–protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure‐based computational methods to optimize the binding affinity of an antibody fragment to the I‐domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd ∼7 nM) and the moderate resolution (2.8 Å) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high‐affinity quadruple mutant complex at 2.2 Å. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher‐affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single‐mutant success rate. Our results indicate that structure‐based computational design can be successfully applied to further improve the binding of high‐affinity antibodies.


The Journal of Infectious Diseases | 2011

Progressive Multifocal Leukoencephalopathy (PML) Development Is Associated With Mutations in JC Virus Capsid Protein VP1 That Change Its Receptor Specificity

Leonid Gorelik; Carl Reid; Manuela Testa; Margot Brickelmaier; Simona Bossolasco; Annamaria Pazzi; Arabella Bestetti; Paul Carmillo; Ewa Wilson; Michele McAuliffe; Christopher J. Tonkin; John P. Carulli; Alexey Lugovskoy; Adriano Lazzarin; Shamil R. Sunyaev; Kenneth J. Simon; Paola Cinque

Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid-independent binding with brain cells.


PLOS Genetics | 2009

Adaptive Mutations in the JC Virus Protein Capsid Are Associated with Progressive Multifocal Leukoencephalopathy (PML)

Shamil R. Sunyaev; Alexey Lugovskoy; Kenneth J. Simon; Leonid Gorelik

PML is a progressive and mostly fatal demyelinating disease caused by JC virus infection and destruction of infected oligodendrocytes in multiple brain foci of susceptible individuals. While JC virus is highly prevalent in the human population, PML is a rare disease that exclusively afflicts only a small percentage of immunocompromised individuals including those affected by HIV (AIDS) or immunosuppressive drugs. Viral- and/or host-specific factors, and not simply immune status, must be at play to account for the very large discrepancy between viral prevalence and low disease incidence. Here, we show that several amino acids on the surface of the JC virus capsid protein VP1 display accelerated evolution in viral sequences isolated from PML patients but not in sequences isolated from healthy subjects. We provide strong evidence that at least some of these mutations are involved in binding of sialic acid, a known receptor for the JC virus. Using statistical methods of molecular evolution, we performed a comprehensive analysis of JC virus VP1 sequences isolated from 55 PML patients and 253 sequences isolated from the urine of healthy individuals and found that a subset of amino acids found exclusively among PML VP1 sequences is acquired via adaptive evolution. By modeling of the 3-D structure of the JC virus capsid, we showed that these residues are located within the sialic acid binding site, a JC virus receptor for cell infection. Finally, we go on to demonstrate the involvement of some of these sites in receptor binding by demonstrating a profound reduction in hemagglutination properties of viral-like particles made of the VP1 protein carrying these mutations. Collectively, these results suggest that a more virulent PML causing phenotype of JC virus is acquired via adaptive evolution that changes viral specificity for its cellular receptor(s).


Bioorganic & Medicinal Chemistry | 2013

Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism.

Douglas Marcotte; Weike Zeng; Jean-Christophe Hus; Andres McKenzie; Cathy Hession; Ping Jin; Chris Bergeron; Alexey Lugovskoy; Istvan Enyedy; Hernan Cuervo; Deping Wang; Cédric Atmanene; Dominique Roecklin; Malgorzata M. Vecchi; Valérie Vivat; Joachim Kraemer; Dirk Winkler; Victor Hong; Jianhua Chao; Matvey E. Lukashev; Laura Silvian

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.


mAbs | 2009

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Jennifer S. Michaelson; Stephen J. Demarest; Brian Robert Miller; Aldo Amatucci; William B. Snyder; Xiufeng Wu; Flora Huang; Samantha Phan; Sharon X. Gao; Adam Doern; Graham K. Farrington; Alexey Lugovskoy; Ingrid Joseph; Veronique Bailly; Xin Wang; Ellen Garber; Jeffrey L. Browning; Scott Glaser

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTβR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti- LTβR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a full-length anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.


Protein Engineering Design & Selection | 2010

Stability engineering of scFvs for the development of bispecific and multivalent antibodies

Brian Robert Miller; Stephen J. Demarest; Alexey Lugovskoy; Flora Huang; Xiufeng Wu; William B. Snyder; Lisa J. Croner; Norman Wang; Aldo Amatucci; Jennifer S. Michaelson; Scott Glaser

Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.


Structure | 2008

Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site.

Mia Rushe; Laura Silvian; Sarah A. Bixler; Ling Ling Chen; Anne Cheung; Scott Bowes; Hernan Cuervo; Steven A. Berkowitz; Timothy S. Zheng; Kevin Guckian; Maria Pellegrini; Alexey Lugovskoy

The phosphorylation of IkappaB by the IKK complex targets it for degradation and releases NF-kappaB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKalpha/beta kinases and a regulatory protein, NF-kappaB essential modulator (NEMO; IKKgamma). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complexs catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.


Molecular Cancer Therapeutics | 2014

MM-141, an IGF-IR– and ErbB3-Directed Bispecific Antibody, Overcomes Network Adaptations That Limit Activity of IGF-IR Inhibitors

Jonathan Fitzgerald; Bryan Johnson; Jason Baum; Sharlene Adams; Sergio Iadevaia; Jian Tang; Victoria Rimkunas; Lihui Xu; Neeraj Kohli; Rachel Rennard; Maja Razlog; Yang Jiao; Brian Harms; Kenneth J. Olivier; Birgit Schoeberl; Ulrik Nielsen; Alexey Lugovskoy

Although inhibition of the insulin-like growth factor (IGF) signaling pathway was expected to eliminate a key resistance mechanism for EGF receptor (EGFR)-driven cancers, the effectiveness of IGF-I receptor (IGF-IR) inhibitors in clinical trials has been limited. A multiplicity of survival mechanisms are available to cancer cells. Both IGF-IR and the ErbB3 receptor activate the PI3K/AKT/mTOR axis, but ErbB3 has only recently been pursued as a therapeutic target. We show that coactivation of the ErbB3 pathway is prevalent in a majority of cell lines responsive to IGF ligands and antagonizes IGF-IR–mediated growth inhibition. Blockade of the redundant IGF-IR and ErbB3 survival pathways and downstream resistance mechanisms was achieved with MM-141, a tetravalent bispecific antibody antagonist of IGF-IR and ErbB3. MM-141 potency was superior to monospecific and combination antibody therapies and was insensitive to variation in the ratio of IGF-IR and ErbB3 receptors. MM-141 enhanced the biologic impact of receptor inhibition in vivo as a monotherapy and in combination with the mTOR inhibitor everolimus, gemcitabine, or docetaxel, through blockade of IGF-IR and ErbB3 signaling and prevention of PI3K/AKT/mTOR network adaptation. Mol Cancer Ther; 13(2); 410–25. ©2013 AACR.


Protein Science | 2010

Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis.

R. Blake Pepinsky; Laura Silvian; Steven A. Berkowitz; Graham K. Farrington; Alexey Lugovskoy; Lee Walus; John K. Eldredge; Allan D. Capili; Sha Mi; Christilyn Graff; Ellen Garber

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti‐LINGO‐1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.

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Neeraj Kohli

Brigham and Women's Hospital

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Ulrik Nielsen

University of California

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Ellen Garber

Northwestern University

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