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Nature Chemical Biology Dec 2021Small molecule drugs form the backbone of modern medicine's therapeutic arsenal. Often less appreciated is the role that small molecules have had in advancing basic... (Review)
Review
Small molecule drugs form the backbone of modern medicine's therapeutic arsenal. Often less appreciated is the role that small molecules have had in advancing basic biology. In this Review, we highlight how resistance mutations have unlocked the potential of small molecule chemical probes to discover new biology. We describe key instances in which resistance mutations and related genetic variants yielded foundational biological insight and categorize these examples on the basis of their role in the discovery of novel molecular mechanisms, protein allostery, physiology and cell signaling. Next, we suggest ways in which emerging technologies can be leveraged to systematically introduce and characterize resistance mutations to catalyze basic biology research and drug discovery. By recognizing how resistance mutations have propelled biological discovery, we can better harness new technologies and maximize the potential of small molecules to advance our understanding of biology and improve human health.
Topics: Alleles; Animals; Diazepam; Drug Discovery; Drug Resistance; Humans; Mutant Proteins; Mutation; Pharmaceutical Preparations; Protein Binding; Protein Conformation; Signal Transduction; Sulfonamides
PubMed: 34799733
DOI: 10.1038/s41589-021-00865-9 -
International Journal of Molecular... Mar 2020Trials using antisense oligonucleotide technology to lower Huntingtin levels in Huntington's disease (HD) are currently ongoing. This progress, taking place only 27... (Review)
Review
Trials using antisense oligonucleotide technology to lower Huntingtin levels in Huntington's disease (HD) are currently ongoing. This progress, taking place only 27 years after the identification of the Huntingtin gene () in 1993 reflects the enormous development in genetic engineering in the last decades. It is also the result of passionate basic scientific work and large worldwide registry studies that have advanced the understanding of HD. Increased knowledge of the pathophysiology of this autosomal dominantly inherited CAG-repeat expansion mediated neurodegenerative disease has led to the development of several putative treatment strategies, currently under investigation. These strategies span the whole spectrum of potential targets from genome editing via RNA interference to promoting protein degradation. Yet, recent studies revealed the importance of huntingtin RNA in the pathogenesis of the disease. Therefore, huntingtin-lowering by means of RNA interference appears to be a particular promising strategy. As a matter of fact, these approaches have entered, or are on the verge of entering, the clinical trial period. Here, we provide an overview of huntingtin-lowering approaches via DNA or RNA interference in present clinical trials as well as strategies subject to upcoming therapeutic options. We furthermore discuss putative implications for future treatment of HD patients.
Topics: Animals; Gene Expression Regulation; Genetic Engineering; Humans; Huntingtin Protein; Huntington Disease; Models, Biological; Mutant Proteins
PubMed: 32245050
DOI: 10.3390/ijms21062146 -
Cells May 2023Amyotrophic lateral sclerosis (ALS) is a neuronal degenerative condition identified via a build-up of mutant aberrantly folded proteins. The native folding of... (Review)
Review
Amyotrophic lateral sclerosis (ALS) is a neuronal degenerative condition identified via a build-up of mutant aberrantly folded proteins. The native folding of polypeptides is mediated by molecular chaperones, preventing their pathogenic aggregation. The mutant protein expression in ALS is linked with the entrapment and depletion of chaperone capacity. The lack of a thorough understanding of chaperones' involvement in ALS pathogenesis presents a significant challenge in its treatment. Here, we review how the accumulation of the ALS-linked mutant FUS, TDP-43, SOD1, and C9orf72 proteins damage cellular homeostasis mechanisms leading to neuronal loss. Further, we discuss how the HSP70 and DNAJ family co-chaperones can act as potential targets for reducing misfolded protein accumulation in ALS. Moreover, small HSPB1 and HSPB8 chaperones can facilitate neuroprotection and prevent stress-associated misfolded protein apoptosis. Designing therapeutic strategies by pharmacologically enhancing cellular chaperone capacity to reduce mutant protein proteotoxic effects on ALS pathomechanisms can be a considerable advancement. Chaperones, apart from directly interacting with misfolded proteins for protein quality control, can also filter their toxicity by initiating strong stress-response pathways, modulating transcriptional expression profiles, and promoting anti-apoptotic functions. Overall, these properties of chaperones make them an attractive target for gaining fundamental insights into misfolded protein disorders and designing more effective therapies against ALS.
Topics: Humans; Amyotrophic Lateral Sclerosis; Proteostasis; Molecular Chaperones; HSP40 Heat-Shock Proteins; Mutant Proteins
PubMed: 37174703
DOI: 10.3390/cells12091302 -
Science (New York, N.Y.) Sep 2023Machine-learning algorithm uses structure prediction to spot disease-causing mutations.
Machine-learning algorithm uses structure prediction to spot disease-causing mutations.
Topics: Algorithms; Machine Learning; Mutant Proteins; Protein Conformation; Genetic Variation; Mutation, Missense; Disease; Sequence Analysis, DNA
PubMed: 37725046
DOI: 10.1126/science.adj8672 -
Biomolecules Apr 2020RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various...
RUVBL1 and RUVBL2 are highly conserved ATPases that belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily and are involved in various complexes and cellular processes, several of which are closely linked to oncogenesis. The proteins were implicated in DNA damage signaling and repair, chromatin remodeling, telomerase activity, and in modulating the transcriptional activities of proto-oncogenes such as c-Myc and β-catenin. Moreover, both proteins were found to be overexpressed in several different types of cancers such as breast, lung, kidney, bladder, and leukemia. Given their various roles and strong involvement in carcinogenesis, the RUVBL proteins are considered to be novel targets for the discovery and development of therapeutic cancer drugs. Here, we describe the identification of sorafenib as a novel inhibitor of the ATPase activity of human RUVBL2. Enzyme kinetics and surface plasmon resonance experiments revealed that sorafenib is a weak, mixed non-competitive inhibitor of the protein's ATPase activity. Size exclusion chromatography and small angle X-ray scattering data indicated that the interaction of sorafenib with RUVBL2 does not cause a significant effect on the solution conformation of the protein; however, the data suggested that the effect of sorafenib on RUVBL2 activity is mediated by the insertion domain in the protein. Sorafenib also inhibited the ATPase activity of the RUVBL1/2 complex. Hence, we propose that sorafenib could be further optimized to be a potent inhibitor of the RUVBL proteins.
Topics: ATPases Associated with Diverse Cellular Activities; Carrier Proteins; DNA Helicases; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Mutant Proteins; Protein Aggregates; Protein Multimerization; Sorafenib
PubMed: 32295120
DOI: 10.3390/biom10040605 -
Journal of Huntington's Disease 2021Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously...
BACKGROUND
Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the huntingtin gene, resulting in a polyglutamine expansion in the ubiquitously expressed mutant huntingtin protein.
OBJECTIVE
Here we set out to identify proteins interacting with the full-length wild-type and mutant huntingtin protein in the mice cortex brain region to understand affected biological processes in Huntington's disease pathology.
METHODS
Full-length huntingtin with 20 and 140 polyQ repeats were formaldehyde-crosslinked and isolated via their N-terminal Flag-tag from 2-month-old mice brain cortex. Interacting proteins were identified and quantified by label-free liquid chromatography-mass spectrometry (LC-MS/MS).
RESULTS
We identified 30 interactors specific for wild-type huntingtin, 14 interactors specific for mutant huntingtin and 14 shared interactors that interacted with both wild-type and mutant huntingtin, including known interactors such as F8a1/Hap40. Syt1, Ykt6, and Snap47, involved in vesicle transport and exocytosis, were among the proteins that interacted specifically with wild-type huntingtin. Various other proteins involved in energy metabolism and mitochondria were also found to associate predominantly with wild-type huntingtin, whereas mutant huntingtin interacted with proteins involved in translation including Mapk3, Eif3h and Eef1a2.
CONCLUSION
Here we identified both shared and specific interactors of wild-type and mutant huntingtin, which are involved in different biological processes including exocytosis, vesicle transport, translation and metabolism. These findings contribute to the understanding of the roles that wild-type and mutant huntingtin play in a variety of cellular processes both in healthy conditions and Huntington's disease pathology.
Topics: Animals; Brain; Chromatography, Liquid; Huntingtin Protein; Huntington Disease; Immunoprecipitation; Mice; Mutant Proteins; Nerve Tissue Proteins; Synaptotagmin I; Tandem Mass Spectrometry
PubMed: 34151850
DOI: 10.3233/JHD-210476 -
Journal of Molecular Biology Oct 2023The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In...
The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In particular, understanding the thermodynamics and kinetics of the folding process is important for uncovering the mechanisms behind human disorders caused by protein misfolding. To address this issue, it is essential to collect and curate experimental kinetic and thermodynamic data on protein folding. K-Pro is a new database designed for collecting and storing experimental kinetic data on monomeric proteins, with a two-state folding mechanism. With 1,529 records from 62 proteins corresponding to 65 structures, K-Pro contains various kinetic parameters such as the logarithm of the folding and unfolding rates, Tanford's β and the ϕ values. When available, the database also includes thermodynamic parameters associated with the kinetic data. K-Pro features a user-friendly interface that allows browsing and downloading kinetic data of interest. The graphical interface provides a visual representation of the protein and mutants, and it is cross-linked to key databases such as PDB, UniProt, and PubMed. K-Pro is open and freely accessible through https://folding.biofold.org/k-pro and supports the latest versions of popular browsers.
Topics: Humans; Databases, Protein; Kinetics; Protein Denaturation; Protein Folding; Proteins; Thermodynamics; Mutant Proteins
PubMed: 37625584
DOI: 10.1016/j.jmb.2023.168245 -
Cell Reports Aug 2023Prion-like protein propagation is considered a common pathogenic mechanism in neurodegenerative diseases. Here we investigate the in vivo propagation pattern and...
Prion-like protein propagation is considered a common pathogenic mechanism in neurodegenerative diseases. Here we investigate the in vivo propagation pattern and aggregation state of mutant α-synuclein by injecting adeno-associated viral (AAV)-α-synuclein-A53T-EGFP into the mouse olfactory cortex. Comparison of aggregation states in various brain regions at multiple time points after injection using western blot analyses shows that the monomeric state of the mutant/misfolded protein propagates to remote brain regions by 2 weeks and that the propagated proteins aggregate in situ after being incorporated into neurons. Moreover, injection of Alexa 488-labeled α-synuclein-A53T confirms the monomeric propagation at 2 weeks. Super-resolution microscopy shows that both α-synuclein-A53T proteins propagate via the lymphatic system, penetrate perineuronal nets, and reach the surface of neurons. Electron microscopy shows that the propagated mutant/misfolded monomer forms fibrils characteristic of Parkinson's disease after its incorporation into neurons. These findings suggest a mode of propagation different from that of aggregate-dependent propagation.
Topics: Animals; Mice; alpha-Synuclein; Brain; Parkinson Disease; Lymphatic System; Blotting, Western; Mutant Proteins
PubMed: 37591248
DOI: 10.1016/j.celrep.2023.112962 -
Nature Apr 2021The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein and is...
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.
Topics: Adenosine Monophosphate; Aged; Alanine; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Chronic Disease; Evolution, Molecular; Genome, Viral; High-Throughput Nucleotide Sequencing; Humans; Immune Evasion; Immune Tolerance; Immunization, Passive; Immunosuppression Therapy; Male; Mutagenesis; Mutant Proteins; Mutation; Phylogeny; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Time Factors; Viral Load; Virus Shedding; COVID-19 Drug Treatment; COVID-19 Serotherapy
PubMed: 33545711
DOI: 10.1038/s41586-021-03291-y -
Acta Pharmacologica Sinica Oct 2022Transcription factors (TFs) specifically bind to DNA, recruit cofactor proteins and modulate target gene expression, rendering them essential roles in the regulation of... (Review)
Review
Transcription factors (TFs) specifically bind to DNA, recruit cofactor proteins and modulate target gene expression, rendering them essential roles in the regulation of numerous biological processes. Meanwhile, mutated or dysregulated TFs are involved in a variety of human diseases. As multiple signaling pathways ultimately converge at TFs, targeting these TFs directly may prove to be more specific and cause fewer side effects, than targeting the upfront conventional targets in these pathways. All these features together endue TFs with great potential and high selectivity as therapeutic drug targets. However, TFs have been historically considered "undruggable", mainly due to their lack of structural information, especially about the appropriate ligand-binding sites and protein-protein interactions, leading to relatively limited choices in the TF-targeting drug design. In this review, we summarize the recent progress of TF-targeting drugs and highlight certain strategies used for targeting TFs, with a number of representative drugs that have been approved or in the clinical trials as examples. Various approaches in targeting TFs directly or indirectly have been developed. Common direct strategies include aiming at defined binding pockets, proteolysis-targeting chimaera (PROTAC), and mutant protein reactivation. In contrast, the indirect ones comprise inhibition of protein-protein interactions between TF and other proteins, blockade of TF expression, targeting the post-translational modifications, and targeting the TF-DNA interactions. With more comprehensive structural information about TFs revealed by the powerful cryo-electron microscopy technology and predicted by machine-learning algorithms, plus more efficient compound screening platforms and a deeper understanding of TF-disease relationships, the development of TF-targeting drugs will certainly be accelerated in the near future.
Topics: Cryoelectron Microscopy; DNA; Humans; Ligands; Mutant Proteins; Transcription Factors
PubMed: 35132191
DOI: 10.1038/s41401-021-00852-9