ABSTRACT
Two of the big challenges in modern bioprocesses are process economics and in-depth process understanding. Getting access to online process data helps to understand process dynamics and monitor critical process parameters (CPPs). This is an important part of the quality-by- design concept that was introduced to the pharmaceutical industry in the last decade. Raman spectroscopy has proven to be a versatile tool to allow noninvasive measurements and access to a broad spectrum of analytes. This information can then be used for enhanced process control strategies. This review article will focus on the latest applications of Raman spectroscopy in established protein production bioprocesses as well as show its potential for virus, cell therapy, and mRNA processes.
Subject(s)
Biological Products , Spectrum Analysis, Raman , Spectrum Analysis, Raman/methods , Biological Products/analysisABSTRACT
The treatment of tumors driven by overexpression or amplification of MYC oncogenes remains a significant challenge in drug discovery. Here, we present a new strategy toward the inhibition of MYC via the disruption of the protein-protein interaction between MYC and its chromatin cofactor WD Repeat-Containing Protein 5. Blocking the association of these proteins is hypothesized to disrupt the localization of MYC to chromatin, thus disrupting the ability of MYC to sustain tumorigenesis. Utilizing a high-throughput screening campaign and subsequent structure-guided design, we identify small-molecule inhibitors of this interaction with potent in vitro binding affinity and report structurally related negative controls that can be used to study the effect of this disruption. Our work suggests that disruption of this protein-protein interaction may provide a path toward an effective approach for the treatment of multiple tumors and anticipate that the molecules disclosed can be used as starting points for future efforts toward compounds with improved drug-like properties.
Subject(s)
Drug Discovery , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Interaction Domains and Motifs/drug effects , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Salicylic Acid/chemistry , Small Molecule Libraries/pharmacology , Sulfonamides/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , HEK293 Cells , High-Throughput Screening Assays , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Protein Binding , Protein Conformation , Proto-Oncogene Proteins c-myc/metabolism , WD40 RepeatsABSTRACT
WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5 H-pyrrolo[1,2- a]imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer.
Subject(s)
Drug Design , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/chemistry , Imidazoles/chemistry , Imidazoles/pharmacology , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Histone-Lysine N-Methyltransferase/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Structure-Activity RelationshipABSTRACT
Negative allosteric modulators (NAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) have potential applications in the treatment of fragile X syndrome, levodopa-induced dyskinesia in Parkinson disease, Alzheimer disease, addiction, and anxiety; however, clinical and preclinical studies raise concerns that complete blockade of mGlu5 and inverse agonist activity of current mGlu5 NAMs contribute to adverse effects that limit the therapeutic use of these compounds. We report the discovery and characterization of a novel mGlu5 NAM, N,N-diethyl-5-((3-fluorophenyl)ethynyl)picolinamide (VU0477573) that binds to the same allosteric site as the prototypical mGlu5 NAM MPEP but displays weak negative cooperativity. Because of this weak cooperativity, VU0477573 acts as a "partial NAM" so that full occupancy of the MPEP site does not completely inhibit maximal effects of mGlu5 agonists on intracellular calcium mobilization, inositol phosphate (IP) accumulation, or inhibition of synaptic transmission at the hippocampal Schaffer collateral-CA1 synapse. Unlike previous mGlu5 NAMs, VU0477573 displays no inverse agonist activity assessed using measures of effects on basal [(3)H]inositol phosphate (IP) accumulation. VU0477573 acts as a full NAM when measuring effects on mGlu5-mediated extracellular signal-related kinases 1/2 phosphorylation, which may indicate functional bias. VU0477573 exhibits an excellent pharmacokinetic profile and good brain penetration in rodents and provides dose-dependent full mGlu5 occupancy in the central nervous system (CNS) with systemic administration. Interestingly, VU0477573 shows robust efficacy, comparable to the mGlu5 NAM MTEP, in models of anxiolytic activity at doses that provide full CNS occupancy of mGlu5 and demonstrate an excellent CNS occupancy-efficacy relationship. VU0477573 provides an exciting new tool to investigate the efficacy of partial NAMs in animal models.