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1.
Mol Pharm ; 18(3): 1048-1060, 2021 03 01.
Article in English | MEDLINE | ID: mdl-33444501

ABSTRACT

Targeted extrahepatic delivery of siRNA remains a challenging task in the field of nucleic acid therapeutics. An ideal delivery tool must internalize siRNA exclusively into the cells of interest without affecting the silencing activity of siRNA. Here, we report the use of anti-EGFR Nanobodies (trademark of Ablynx N.V.) as tools for targeted siRNA delivery. A straightforward procedure for site-specific conjugation of siRNA to an engineered C-terminal cysteine residue on the Nanobody (trademark of Ablynx N.V.) is described. We show that siRNA-conjugated Nanobodies (Nb-siRNA) retain their binding to EGFR and enter EGFR-positive cells via receptor-mediated endocytosis. The activity of Nb-siRNAs was assessed by measuring the knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells. We demonstrate that Nb-siRNAs are active in vitro and induce mRNA cleavage in the targeted cell line. In addition, we discuss the silencing activity of siRNA conjugated to fused Nbs with various combinations of EGFR-binding building blocks. Finally, we compare the performance of Nb-siRNA joined by four different linkers and discuss the advantages and limitations of using cleavable and noncleavable linkers in the context of Nanobody-mediated siRNA delivery.


Subject(s)
Neoplasms/genetics , Neoplasms/therapy , RNA, Small Interfering/genetics , Single-Domain Antibodies/genetics , Cell Line, Tumor , ErbB Receptors/genetics , Gene Silencing/physiology , Hep G2 Cells , Humans , Nucleic Acids/genetics
2.
Front Immunol ; 8: 884, 2017.
Article in English | MEDLINE | ID: mdl-28871249

ABSTRACT

The heterodimeric cytokine interleukin (IL) 23 comprises the IL12-shared p40 subunit and an IL23-specific subunit, p19. Together with IL12 and IL27, IL23 sits at the apex of the regulatory mechanisms shaping adaptive immune responses. IL23, together with IL17, plays an important role in the development of chronic inflammation and autoimmune inflammatory diseases. In this context, we generated monovalent antihuman IL23 variable heavy chain domain of llama heavy chain antibody (VHH) domains (Nanobodies®) with low nanomolar affinity for human interleukin (hIL) 23. The crystal structure of a quaternary complex assembling hIL23 and several nanobodies against p19 and p40 subunits allowed identification of distinct epitopes and enabled rational design of a multivalent IL23-specific blocking nanobody. Taking advantage of the ease of nanobody formatting, multivalent IL23 nanobodies were assembled with properly designed linkers flanking an antihuman serum albumin nanobody, with improved hIL23 neutralization capacity in vitro and in vivo, as compared to the monovalent nanobodies. These constructs with long exposure time are excellent candidates for further developments targeting Crohn's disease, rheumatoid arthritis, and psoriasis.

3.
Curr Top Med Chem ; 15(24): 2543-57, 2015.
Article in English | MEDLINE | ID: mdl-26126902

ABSTRACT

Nanobodies are therapeutic proteins derived from the variable domain (VHH) of naturally occurring heavy-chain antibodies. These VHH domains are the smallest functional fragments derived from a naturally occurring immunoglobulin. Nanobodies can be easily produced in prokaryotic or eukaryotic host organisms and their unique biophysical characteristics render these molecules ideal candidates for drug development. They are also emerging as an interesting new class of potential therapeutics for targets such as GPCRs, which have historically been challenging for small molecule drug discovery and even more difficult for biologics discovery. The ability to easily combine Nanobodies with different binding sites and different modes of action can be used to generate highly selective and highly potent drug candidates with very attractive pharmacological profiles. In addition, Nanobodies have been used as crystallization chaperones to enable or facilitate the structural determination of an active GPCR conformation.


Subject(s)
Drug Discovery/methods , Receptors, G-Protein-Coupled/metabolism , Single-Domain Antibodies/metabolism , Animals , Binding Sites , Humans
5.
Bioorg Med Chem Lett ; 19(9): 2492-6, 2009 May 01.
Article in English | MEDLINE | ID: mdl-19342234

ABSTRACT

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC(50)=400nM and 270nM, respectively) and selective (CC(50)>20muM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.


Subject(s)
Antiviral Agents/chemical synthesis , Benzodiazepines/chemistry , Chemistry, Pharmaceutical/methods , Hepacivirus/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Acrylates/chemistry , Antiviral Agents/pharmacology , Crystallography, X-Ray , Drug Design , Hepacivirus/enzymology , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Structure , Structure-Activity Relationship
6.
Antimicrob Agents Chemother ; 52(12): 4420-31, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18852280

ABSTRACT

The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.


Subject(s)
Benzodiazepines/pharmacology , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Benzodiazepines/chemistry , Benzodiazepines/metabolism , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Genotype , Hepacivirus/enzymology , Hepacivirus/genetics , Hepacivirus/physiology , Humans , Models, Molecular , Molecular Sequence Data , Mutation , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
7.
Bioorg Med Chem Lett ; 18(17): 4853-8, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18678486

ABSTRACT

SAR analysis performed with a limited set of cyclopentane-containing macrocycles led to the identification of N-[17-[2-(4-isopropylthiazole-2-yl)-7-methoxy-8-methylquinolin-4-yloxy]-13-methyl-2,14-dioxo-3,13-diazatricyclo [13.3.0.0(4,6)]octadec-7-ene-4-carbonyl](cyclopropyl)sulfonamide (TMC435350, 32c) as a potent inhibitor of HCV NS3/4A protease (K(i)=0.36nM) and viral replication (replicon EC(50)=7.8nM). TMC435350 also displayed low in vitro clearance and high permeability, which were confirmed by in vivo pharmacokinetic studies. TMC435350 is currently being evaluated in the clinics.


Subject(s)
Carrier Proteins/antagonists & inhibitors , Cyclopentanes/pharmacology , Hepacivirus/drug effects , Hepacivirus/enzymology , Heterocyclic Compounds, 3-Ring/pharmacology , Macrocyclic Compounds/pharmacology , Protease Inhibitors/pharmacology , Sulfonamides/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Animals , Caco-2 Cells , Cell Line , Cyclopentanes/chemistry , Dogs , Hepatitis C/drug therapy , Heterocyclic Compounds, 3-Ring/chemistry , Humans , Intracellular Signaling Peptides and Proteins , Macrocyclic Compounds/chemistry , Male , Protease Inhibitors/chemistry , Rats , Rats, Sprague-Dawley , Simeprevir , Structure-Activity Relationship , Sulfonamides/chemistry
8.
J Virol ; 81(13): 6909-19, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17459932

ABSTRACT

The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a.


Subject(s)
Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , Pyrrolidines/chemistry , RNA-Dependent RNA Polymerase/chemistry , Amino Acid Substitution , Base Sequence , Binding Sites/genetics , Genotype , Hepacivirus/genetics , Humans , Molecular Sequence Data , Mutation, Missense , Protein Structure, Tertiary , RNA-Dependent RNA Polymerase/genetics
9.
J Med Chem ; 48(6): 2115-20, 2005 Mar 24.
Article in English | MEDLINE | ID: mdl-15771454

ABSTRACT

The development of drug-resistant viruses limits the therapeutic success of anti-HIV therapies. Some of these genetic HIV-variants display complex mutational patterns in their pol gene that codes for protease and reverse transcriptase, the most investigated molecular targets for antiretroviral therapy. In this paper, we present a computational structure-based approach to predict the resistance of a HIV-1 protease strain to amprenavir by calculating the interaction energy of the drug with HIV-1 protease. By considering the interaction energy per residue, we can identify what residue mutations contribute to drug-resistance. This approach is presented here as a structure-based tool for the prediction of resistance of HIV-1 protease toward amprenavir, with a view to use the drug-protein interaction-energy pattern in a lead-optimization procedure for the discovery of new anti-HIV drugs.


Subject(s)
HIV Protease Inhibitors/chemistry , HIV Protease/chemistry , HIV Protease/genetics , Quantitative Structure-Activity Relationship , Carbamates , Drug Resistance, Viral , Furans , Genotype , HIV-1/drug effects , Mutation , Sulfonamides/chemistry , Sulfonamides/pharmacology , Thermodynamics
10.
J Virol ; 76(15): 7595-606, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12097573

ABSTRACT

It has been proposed that the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 (e-gp41), involved in HIV entry into the target cell, exists in at least two conformations, a pre-hairpin intermediate and a fusion-active hairpin structure. To obtain more information on the structure-sequence relationship in e-gp41, we performed in silico a full single-amino-acid substitution analysis, resulting in a Fold Compatible Database (FCD) for each conformation. The FCD contains for each residue position in a given protein a list of values assessing the energetic compatibility (ECO) of each of the 20 natural amino acids at that position. Our results suggest that FCD predictions are in good agreement with the sequence variation observed for well-validated e-gp41 sequences. The data show that at a minECO threshold value of 5 kcal/mol, about 90% of the observed patient sequence variation is encompassed by the FCD predictions. Some inconsistent FCD predictions at N-helix positions packing against residues of the C helix suggest that packing of both peptides may involve some flexibility and may be attributed to an altered orientation of the C-helical domain versus the N-helical region. The permissiveness of sequence variation in the C helices is in agreement with FCD predictions. Comparison of N-core and triple-hairpin FCDs suggests that the N helices may impose more constraints on sequence variation than the C helices. Although the observed sequences of e-gp41 contain many multiple mutations, our method, which is based on single-point mutations, can predict the natural sequence variability of e-gp41 very well.


Subject(s)
Amino Acid Sequence , Genetic Variation , HIV Envelope Protein gp41/genetics , HIV-1/classification , Protein Conformation , Amino Acid Substitution , Databases, Factual , Genotype , HIV Envelope Protein gp41/chemistry , HIV Infections/virology , HIV-1/genetics , HIV-1/isolation & purification , Humans , Membrane Fusion , Molecular Sequence Data , Phenotype , Protein Folding
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