ABSTRACT
Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.
Subject(s)
Antineoplastic Agents , Immunoconjugates , Animals , Antibodies , Antineoplastic Agents/therapeutic use , CHO Cells , Cricetinae , Cricetulus , Cysteine , Disulfides , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Metabolic EngineeringABSTRACT
Over the past decade, single-use tangential flow filtration (TFF) technologies have emerged to reduce system preparation time, promote fast and flexible product change over, and ultimately shorten process development and manufacturing time/cost. In this study, the performance of a recently developed Pellicon® single-use TFF capsule was compared against traditional Pellicon® cassettes by assessing TFF process performance (such as flux, residuals clearance, and yield) and post-purification product attributes (such as concentration and mass-weighted average molecular weight). Good scaling was shown by comparing process performance and product attributes across different scales and formats. Additionally, similar TFF process performance and post-purification product attributes were observed for the single-use capsule compared to the reusable TFF cassettes. The capsule requires a smaller flush than the cassette, and it is easier to use since it does not require a compression holder or pre-sanitization. The results provide insight into the application of the single-use TFF capsule and scalability of TFF processes for the purification of conjugate vaccines.
Subject(s)
Filtration/methods , Polysaccharides , Vaccines, Conjugate/chemistry , Biotechnology/methods , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Polysaccharides/metabolismABSTRACT
Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems.
Subject(s)
Antibodies , Cysteine , Protein Engineering/methods , Recombinant Proteins , Animals , Antibodies/chemistry , Antibodies/isolation & purification , Antibodies/metabolism , CHO Cells , Cricetinae , Cricetulus , Cysteine/chemistry , Cysteine/metabolism , Dinitrobenzenes , HEK293 Cells , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.
Subject(s)
Anti-HIV Agents/chemistry , Enzyme Inhibitors/chemistry , Ribonuclease H, Human Immunodeficiency Virus/antagonists & inhibitors , Thiocarbamates/chemistry , Triazoles/chemistry , Amino Acid Sequence , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Cell Line , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Humans , Molecular Sequence Data , Ribonuclease H, Human Immunodeficiency Virus/metabolism , Structure-Activity Relationship , Thiocarbamates/chemical synthesis , Thiocarbamates/pharmacology , Triazoles/chemical synthesis , Triazoles/pharmacology , Virus Replication/drug effectsABSTRACT
We previously reported that a 3-pyridinecarbonitrile analog with a furan substituent at C-5 and a 4-methylindol-5-ylamino substituent at C-4, 1, was a potent inhibitor of PKCtheta (IC50=4.5 nM). Replacement of the C-5 furan ring of 1 with bicyclic heteroaryl rings, led to compounds with significantly improved potency against PKCtheta. Analog 6b with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-1-benzofuran-2-yl group at C-5 had an IC50 value of 0.28 nM for the inhibition of PKCtheta.
Subject(s)
Aminopyridines/chemistry , Isoenzymes/antagonists & inhibitors , Nitriles/chemistry , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Aminopyridines/chemical synthesis , Aminopyridines/pharmacology , Animals , Benzofurans/chemical synthesis , Benzofurans/chemistry , Benzofurans/pharmacology , Half-Life , Humans , Interleukin-2/metabolism , Isoenzymes/metabolism , Mice , Microsomes, Liver/metabolism , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase C/metabolism , Protein Kinase C-theta , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Rats , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
The key intermediate, 4-chloro-5-iodo-3-pyridinecarbonitrile, allowed for ready optimization of the PKCtheta inhibitory activity of a series of 3-pyridinecarbonitriles. Analog 13b with a 4-methylindol-5-ylamino group at C-4 and a 4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl group at C-5 had an IC(50) value of 7.4nM for the inhibition of PKCtheta.
Subject(s)
Indoles/chemistry , Isoenzymes/antagonists & inhibitors , Nitriles/chemistry , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Animals , Indoles/chemical synthesis , Indoles/pharmacology , Isoenzymes/metabolism , Mice , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase C/metabolism , Protein Kinase C-theta , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Structure-Activity RelationshipABSTRACT
A series of potent anthranilic acid-based inhibitors of the hepatitis C NS5B polymerase has been identified. The inhibitors bind to a site on NS5B between the thumb and palm regions adjacent to the active site as determined by X-ray crystallography of the enzyme-inhibitor complex. Guided by both molecular modeling and traditional SAR, the enzyme activity of the initial hit was improved by approximately 100-fold, yielding a series of potent and selective NS5B inhibitors with IC50 values as low as 10 nM. These compounds were also inhibitors of the HCV replicon in cultured HUH7 cells.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , ortho-Aminobenzoates/chemical synthesis , Allosteric Regulation , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Cell Line , Crystallography, X-Ray , Hepacivirus/enzymology , Hepacivirus/genetics , Humans , Models, Molecular , Replicon , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/pharmacologyABSTRACT
The preparation of alpha-methylbenzyl thioureas and their biological activity against varicella zoster virus is described. Several analogs demonstrated IC50s<0.1 microM and their SAR are discussed. These compounds represent a novel class of potent and selective nonnucleoside inhibitors of varicella zoster virus.
Subject(s)
Antiviral Agents/pharmacology , Herpesvirus 3, Human/drug effects , Thiourea/pharmacology , Antiviral Agents/chemistry , Microbial Sensitivity Tests , Structure-Activity Relationship , Thiourea/chemistryABSTRACT
Class A-class C mechanism-based beta-lactamase inhibitors were designed on the basis of the intermediacy of an oxycarbenium species capable of cross-linking with amino acids residues in the active site. Penams 24 and 27 were very potent against AmpC in vitro. The MIC values of 24 in combination with piperacillin against class A and class C producing organisms showed improvement over clinically used tazobactam.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins , Cyclopropanes/chemical synthesis , Diazonium Compounds/chemistry , Enzyme Inhibitors/chemical synthesis , Rhodium , Serine/chemistry , Spiro Compounds/chemical synthesis , Sulbactam/chemistry , beta-Lactamase Inhibitors , beta-Lactams/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Catalysis , Crystallography, X-Ray , Cyclopropanes/chemistry , Cyclopropanes/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Microbial Sensitivity Tests , Models, Molecular , Pseudomonas aeruginosa/drug effects , Serratia marcescens/drug effects , Spiro Compounds/chemistry , Spiro Compounds/pharmacology , Structure-Activity Relationship , Sulbactam/analogs & derivatives , beta-Lactamases/chemistry , beta-Lactams/chemistry , beta-Lactams/pharmacologyABSTRACT
Resistance to antibiotics is currently a major health concern in treating infectious diseases. The most common mechanism of resistance to beta-lactam antibiotics is the production of beta-lactamases, which destroy beta-lactam antibiotics before they reach the bacterial target. Combination therapy, which involves treatment with a beta-lactam antibiotic and a beta-lactamase inhibitor, has been successfully used to control resistance during last two decades. Due to the lack of effectiveness of the currently available beta-lactamase inhibitors against class C enzymes and new variants of beta-lactamases, there is a need to develop an inhibitor with broad-spectrum activity. Since the discovery of clavulanic acid, there has been an enormous research effort in this area to identify better antibiotic/inhibitor combinations and to understand the molecular bases for interactions between beta-lactam antibiotics, beta-lactamases, and beta-lactamase inhibitors. This review describes some of the structure- and mechanism-based approaches to design of new beta-lactamase inhibitors and the study of probable mechanisms of inhibition using X-ray, electrospray ionization mass spectrometry, and molecular modeling techniques.
