Publications by authors named "Aleksandar Antanasijevic"

27 Publications

  • Page 1 of 1

Enhancing glycan occupancy of soluble HIV-1 envelope trimers to mimic the native viral spike.

Cell Rep 2021 Apr;35(1):108933

Department of Medical Microbiology, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam 1105 AZ, the Netherlands; Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, NY, USA. Electronic address:

Artificial glycan holes on recombinant Env-based vaccines occur when a potential N-linked glycosylation site (PNGS) is under-occupied, but not on their viral counterparts. Native-like SOSIP trimers, including clinical candidates, contain such holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To eliminate glycan holes and mimic the glycosylation of native BG505 Env, we replace all 12 NxS sequons on BG505 SOSIP with NxT. All PNGS, except N133 and N160, are nearly fully occupied. Occupancy of the N133 site is increased by changing N133 to NxS, whereas occupancy of the N160 site is restored by reverting the nearby N156 sequon to NxS. Hence, PNGS in close proximity, such as in the N133-N137 and N156-N160 pairs, affect each other's occupancy. We further apply this approach to improve the occupancy of several Env strains. Increasing glycan occupancy should reduce off-target immune responses to vaccine antigens.
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http://dx.doi.org/10.1016/j.celrep.2021.108933DOI Listing
April 2021

Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.

NPJ Vaccines 2020 Aug 5;5(1):72. Epub 2020 Aug 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
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http://dx.doi.org/10.1038/s41541-020-00223-1DOI Listing
August 2020

Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles.

NPJ Vaccines 2021 Feb 9;6(1):24. Epub 2021 Feb 9.

Department of Medical Microbiology, Amsterdam UMC, University of Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands.

The HIV-1 envelope glycoprotein trimer is poorly immunogenic because it is covered by a dense glycan shield. As a result, recombinant Env glycoproteins generally elicit inadequate antibody levels that neutralize clinically relevant, neutralization-resistant (Tier-2) HIV-1 strains. Multivalent antigen presentation on nanoparticles is an established strategy to increase vaccine-driven immune responses. However, due to nanoparticle instability in vivo, the display of non-native Env structures, and the inaccessibility of many neutralizing antibody (NAb) epitopes, the effects of nanoparticle display are generally modest for Env trimers. Here, we generate two-component self-assembling protein nanoparticles presenting twenty SOSIP trimers of the clade C Tier-2 genotype 16055. We show in a rabbit immunization study that these nanoparticles induce 60-fold higher autologous Tier-2 NAb titers than the corresponding SOSIP trimers. Epitope mapping studies reveal that the presentation of 16055 SOSIP trimers on these nanoparticle focuses antibody responses to an immunodominant apical epitope. Thus, these nanoparticles are a promising platform to improve the immunogenicity of Env trimers with apex-proximate NAb epitopes.
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http://dx.doi.org/10.1038/s41541-021-00285-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873233PMC
February 2021

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate.

Science 2020 11 20;370(6520):1089-1094. Epub 2020 Oct 20.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Vaccine efforts to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the current coronavirus disease 2019 (COVID-19) pandemic, are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. We performed cryo-election microscopy and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax, which is based on a full-length spike protein formulated in polysorbate 80 detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared with published spike ectodomain structures. We also observed interactions between the spike trimers, allowing formation of higher-order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.
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http://dx.doi.org/10.1126/science.abe1502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857404PMC
November 2020

Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.

NPJ Vaccines 2020 5;5:72. Epub 2020 Aug 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 USA.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
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http://dx.doi.org/10.1038/s41541-020-00223-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406516PMC
August 2020

Structural analysis of full-length SARS-CoV-2 spike protein from an advanced vaccine candidate.

bioRxiv 2020 Aug 6. Epub 2020 Aug 6.

Dept. of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

Vaccine efforts against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the current COVID-19 pandemic are focused on SARS-CoV-2 spike glycoprotein, the primary target for neutralizing antibodies. Here, we performed cryo-EM and site-specific glycan analysis of one of the leading subunit vaccine candidates from Novavax based on a full-length spike protein formulated in polysorbate 80 (PS 80) detergent. Our studies reveal a stable prefusion conformation of the spike immunogen with slight differences in the S1 subunit compared to published spike ectodomain structures. Interestingly, we also observed novel interactions between the spike trimers allowing formation of higher order spike complexes. This study confirms the structural integrity of the full-length spike protein immunogen and provides a basis for interpreting immune responses to this multivalent nanoparticle immunogen.
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http://dx.doi.org/10.1101/2020.08.06.234674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418715PMC
August 2020

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.

PLoS Pathog 2020 08 11;16(8):e1008665. Epub 2020 Aug 11.

Department of Integrative, Structural and Computational Biology, Scripps Research, La Jolla, California, United States of America.

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.
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http://dx.doi.org/10.1371/journal.ppat.1008665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418955PMC
August 2020

Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens.

Elife 2020 08 4;9. Epub 2020 Aug 4.

Department of Biochemistry, University of Washington, Seattle, United States.

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.
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http://dx.doi.org/10.7554/eLife.57659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402677PMC
August 2020

Structure of avian influenza hemagglutinin in complex with a small molecule entry inhibitor.

Life Sci Alliance 2020 08 1;3(8). Epub 2020 Jul 1.

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA

HA plays a critical role in influenza infection and, thus HA is a potential target for antivirals. Recently, our laboratories have described a novel fusion inhibitor, termed CBS1117, with EC ∼3 μM against group 1 HA. In this work, we characterize the binding properties of CBS1117 to avian H5 HA by x-ray crystallography, NMR, and mutagenesis. The x-ray structure of the complex shows that the compound binds near the HA fusion peptide, a region that plays a critical role in HA-mediated fusion. NMR studies demonstrate binding of CBS1117 to H5 HA in solution and show extensive hydrophobic contacts between the compound and HA surface. Mutagenesis studies further support the location of the compound binding site proximal to the HA fusion peptide and identify additional amino acids that are important to compound binding. Together, this work gives new insights into the CBS1117 mechanism of action and can be exploited to further optimize this compound and better understand the group specific activity of small-molecule inhibitors of HA-mediated entry.
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http://dx.doi.org/10.26508/lsa.202000724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335401PMC
August 2020

HIV-1 Envelope and MPER Antibody Structures in Lipid Assemblies.

Cell Rep 2020 04;31(4):107583

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Center for HIV/AIDS Vaccine Development, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

Structural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstitute a full-length Env clone into a nanodisc, complex it with a membrane-proximal external region (MPER) targeting antibody 10E8, and structurally define the full quaternary epitope of 10E8 consisting of lipid, MPER, and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapt the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with a peptidisc scaffold.
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http://dx.doi.org/10.1016/j.celrep.2020.107583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196886PMC
April 2020

Identification of entry inhibitors with 4-aminopiperidine scaffold targeting group 1 influenza A virus.

Antiviral Res 2020 05 25;177:104782. Epub 2020 Mar 25.

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. Electronic address:

Influenza A viruses (IAVs) cause seasonal flu and occasionally pandemics. The current therapeutics against IAVs target two viral proteins - neuraminidase (NA) and M2 ion-channel protein. However, M2 ion channel inhibitors (amantadine and rimantadine) are no longer recommended by CDC for use due to the emergence of high level of antiviral resistance among the circulating influenza viruses, and resistant strains to NA inhibitors (oseltamivir and zanamivir) have also been reported. Therefore, development of novel anti-influenza therapies is urgently needed. As one of the viral surface glycoproteins, hemagglutinin (HA) mediates critical virus entry steps including virus binding to host cells and virus-host membrane fusion, which makes it a potential target for anti-influenza drug development. In this study, we report the identification of compound CBS1116 with a 4-aminopiperidine scaffold from a chemical library screen as an entry inhibitor specifically targeting two group 1 influenza A viruses, A/Puerto Rico/8/34 (H1N1) and recombinant low pathogenic avian H5N1 virus (A/Vietnam/1203/04, VN04). Mechanism of action studies show that CBS1116 interferes with the HA-mediated fusion process. Further structure activity relationship study generated a more potent compound CBS1117 which has a 50% inhibitory concentration of 70 nM and a selectivity index of ~4000 against A/Puerto Rico/8/34 (H1N1) infection in human lung epithelial cell line (A549).
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http://dx.doi.org/10.1016/j.antiviral.2020.104782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243365PMC
May 2020

Identification of a pH sensor in Influenza hemagglutinin using X-ray crystallography.

J Struct Biol 2020 01 2;209(1):107412. Epub 2019 Nov 2.

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S Ashland Ave, 60607 Chicago, USA. Electronic address:

Hemagglutnin (HA) mediates entry of influenza virus through a series of conformational changes triggered by the low pH of the endosome. The residue or combination of residues acting as pH sensors has not yet been fully elucidated. In this work, we assay pH effects on the structure of H5 HA by soaking HA crystallized at pH 6.5 in a series of buffers with lower pH, mimicking the conditions of the endosome. We find that HA1-H38, which is conserved in Group 1 HA, undergoes a striking change in side chain conformation, which we attribute to its protonation and cation-cation repulsion with conserved HA1-H18. This work suggests that x-ray crystallography can be applied for studying small-scale pH-induced conformational changes providing valuable information on the location of pH sensors in HA. Importantly, the observed change in HA1-H38 conformation is further evidence that the pH-induced conformational changes of HA are the result of a series of protonation events to conserved and non-conserved pH sensors.
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http://dx.doi.org/10.1016/j.jsb.2019.107412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111647PMC
January 2020

Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle.

Nat Commun 2019 09 19;10(1):4272. Epub 2019 Sep 19.

Amsterdam UMC, Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, University of Amsterdam, Amsterdam, 1105AZ, The Netherlands.

The development of native-like HIV-1 envelope (Env) trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP trimers make these NPs a promising immunogen platform.
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http://dx.doi.org/10.1038/s41467-019-12080-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753213PMC
September 2019

A Novel l-Asparaginase with low l-Glutaminase Coactivity Is Highly Efficacious against Both T- and B-cell Acute Lymphoblastic Leukemias .

Cancer Res 2018 03 17;78(6):1549-1560. Epub 2018 Jan 17.

The Jesse Brown VA Medical Center, Chicago, Illinois.

Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL. A new l-asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-2106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856643PMC
March 2018

Probing the metastable state of influenza hemagglutinin.

J Biol Chem 2017 12 10;292(52):21590-21597. Epub 2017 Nov 10.

From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607

Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.
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http://dx.doi.org/10.1074/jbc.M117.815043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766969PMC
December 2017

Molecular Mechanism Underlying the Action of Influenza A Virus Fusion Inhibitor MBX2546.

ACS Infect Dis 2017 05 3;3(5):330-335. Epub 2017 Apr 3.

Microbiotix Inc., One Innovation Drive, Worcester, Massachusetts 01605, United States.

Influenza A virus envelop protein hemagglutinin (HA) plays important roles in viral entry. We previously have reported that MBX2546, a novel influenza A virus inhibitor, binds to HA and inhibits HA-mediated membrane fusion. In this report, we show that (i) both binding and stabilization of HA by MBX2546 are required for the inhibition of viral infection and (ii) the binding of HA by MBX2546 represses the low-pH-induced conformational change in the HA, which is a prerequisite for membrane fusion. Mutations in MBX2546-resistant influenza A/PR/8/34 (H1N1) viruses are mapped in the HA stem region near the amino terminus of HA2. Finally, we have modeled the binding site of MBX2546 using molecular dynamics and find that the resulting structure is in good agreement with our results. Together, these studies underscore the importance of the HA stem loop region as a potential target for therapeutic intervention.
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http://dx.doi.org/10.1021/acsinfecdis.6b00194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458777PMC
May 2017

A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family.

ACS Chem Biol 2017 05 20;12(5):1245-1256. Epub 2017 Mar 20.

Department of Chemistry, Northwestern University , Evanston, 60208, Illinois, United States.

MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms.
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http://dx.doi.org/10.1021/acschembio.6b01060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652073PMC
May 2017

Stabilization and Improvement of a Promising Influenza Antiviral: Making a PAIN PAINless.

ACS Infect Dis 2016 09 1;2(9):608-615. Epub 2016 Aug 1.

Department of Biochemistry & Molecular Genetics, University of Illinois at Chicago , 900 South Ashland Avenue, Chicago, Illinois 60607, United States.

The viral envelope protein hemagglutinin (HA) plays a critical role in influenza entry and thus is an attractive target for novel therapeutics. The small molecule tert-butylhydroquinone (TBHQ) has previously been shown to bind to HA and inhibit HA-mediated entry with low micromolar potency. However, enthusiasm for the use of TBHQ has diminished due to the compound's antioxidant properties. In this work we show that the antioxidant properties of TBHQ are not responsible for the inhibition of HA-mediated entry. In addition, we have performed a structure-activity relationship (SAR) analysis of TBHQ derivatives. We find that the most promising compound, 3-tert-butyl-4-methoxyphenol, exhibits enhanced potency (IC = 0.6 μM), decreased toxicity (CC = 340 μM), and increased stability (t > 48 h). Finally, we have characterized the binding properties of 3-tert-butyl-4-methoxyphenol using NMR and molecular dynamics to guide future efforts for chemical optimization.
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http://dx.doi.org/10.1021/acsinfecdis.6b00046DOI Listing
September 2016

Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions.

J Biomol NMR 2016 Mar 26;64(3):255-65. Epub 2016 Feb 26.

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S Ashland, Chicago, IL, 60607, USA.

The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.
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http://dx.doi.org/10.1007/s10858-016-0025-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826305PMC
March 2016

Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins.

J Biol Chem 2015 Jul 28;290(27):16595-606. Epub 2015 May 28.

From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607,

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the -6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 β2-β3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated.
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http://dx.doi.org/10.1074/jbc.M115.646695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505413PMC
July 2015

Comparison of the sensitivities of WaterLOGSY and saturation transfer difference NMR experiments.

J Biomol NMR 2014 Sep;60(1):37-44

The WaterLOGSY (WL) and saturation transfer difference (STD) NMR experiments have proven to be extremely useful techniques to characterize interactions between small molecules and large biomolecules. In this work we compare the relative sensitivities of WL and STD NMR using 3 experimental systems: ketoprofen (KET)-bovine serum albumin (BSA), tert-butyl hydroquinone (TBHQ)-hemagglutinin (HA), and chloramphenicol (CAM)-ribosome (70S). In all cases we find that WL is more sensitive than STD for a given experimental time with the ratios ranging from 3.2 for KET-BSA to 16 for TBHQ-HA and CAM-70S. We attribute the increased sensitivity of WL to be due to simultaneous saturation of multiple sources of cross correlation, including direct NOEs of 1H of water and exchangeable groups and indirect NOEs of 1H-C groups. We suggest that the outstanding sensitivity of WL make it ideally suited for drug screening efforts targeting very large biomolecules at relatively low concentrations.
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http://dx.doi.org/10.1007/s10858-014-9848-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201884PMC
September 2014

Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.

J Biol Chem 2014 Aug 19;289(32):22237-45. Epub 2014 Jun 19.

From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607,

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry.
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http://dx.doi.org/10.1074/jbc.M114.572974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139235PMC
August 2014

Ligand screening using NMR.

Methods Mol Biol 2014 ;1140:305-13

Center for Structural Biology, University of Illinois at Chicago, 1100 S. Ashland Ave, Chicago, IL, 60607, USA.

NMR has proven to be an invaluable technique for identifying and characterizing ligand interactions with biomolecules. NMR-based detection of ligand binding to protein targets is described. Specifically, the use of the WaterLOGSY NMR experiment to screen mixtures of compounds from a fragment library for binding to influenza H5 hemagglutinin is detailed.
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http://dx.doi.org/10.1007/978-1-4939-0354-2_22DOI Listing
October 2014

Discovery of selective inhibitors of the Clostridium difficile dehydroquinate dehydratase.

PLoS One 2014 21;9(2):e89356. Epub 2014 Feb 21.

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois, United States of America.

A vibrant and healthy gut flora is essential for preventing the proliferation of Clostridium difficile, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most C. difficile infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a C. difficile specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the C. difficile enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3(rd) of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I C. difficile DHQD but not the type II DHQD from Bacteroides thetaiotaomicron, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the C. difficile enzyme with Ki values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the C. difficile but not the highly homologous Salmonella enterica DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of C. difficile specific antibiotics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089356PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931744PMC
January 2015

Crystal structures of type I dehydroquinate dehydratase in complex with quinate and shikimate suggest a novel mechanism of Schiff base formation.

Biochemistry 2014 Feb 31;53(5):872-80. Epub 2014 Jan 31.

Center for Structural Genomics of Infectious Diseases and Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University , Chicago, Illinois 60611, United States.

A component of the shikimate biosynthetic pathway, dehydroquinate dehydratase (DHQD) catalyzes the dehydration of 3-dehydroquniate (DHQ) to 3-dehydroshikimate. In the type I DHQD reaction mechanism a lysine forms a Schiff base intermediate with DHQ. The Schiff base acts as an electron sink to facilitate the catalytic dehydration. To address the mechanism of Schiff base formation, we determined structures of the Salmonella enterica wild-type DHQD in complex with the substrate analogue quinate and the product analogue shikimate. In addition, we determined the structure of the K170M mutant (Lys170 being the Schiff base forming residue) in complex with quinate. Combined with nuclear magnetic resonance and isothermal titration calorimetry data that revealed altered binding of the analogue to the K170M mutant, these structures suggest a model of Schiff base formation characterized by the dynamic interplay of opposing forces acting on either side of the substrate. On the side distant from the substrate 3-carbonyl group, closure of the enzyme's β8-α8 loop is proposed to guide DHQ into the proximity of the Schiff base-forming Lys170. On the 3-carbonyl side of the substrate, Lys170 sterically alters the position of DHQ's reactive ketone, aligning it at an angle conducive for nucleophilic attack. This study of a type I DHQD reveals the interplay between the enzyme and substrate required for the correct orientation of a functional group constrained within a cyclic substrate.
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http://dx.doi.org/10.1021/bi4015506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985847PMC
February 2014

New small molecule entry inhibitors targeting hemagglutinin-mediated influenza a virus fusion.

J Virol 2014 Feb 6;88(3):1447-60. Epub 2013 Nov 6.

Microbiotix Inc., Worcester, Massachusetts, USA.

Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC(50)] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM(2) % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.
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http://dx.doi.org/10.1128/JVI.01225-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911584PMC
February 2014

Inhibition of influenza H7 hemagglutinin-mediated entry.

PLoS One 2013 23;8(10):e76363. Epub 2013 Oct 23.

Department of Biochemistry & Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois, United States of America.

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50 ~ 6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076363PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806803PMC
February 2015