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1.
Cell ; 187(3): 517-520, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38306978

RESUMO

Structural biology, as powerful as it is, can be misleading. We highlight four fundamental challenges: interpreting raw experimental data; accounting for motion; addressing the misleading nature of in vitro structures; and unraveling interactions between drugs and "anti-targets." Overcoming these challenges will amplify the impact of structural biology on drug discovery.


Assuntos
Descoberta de Drogas , Biologia Molecular , Beleza
2.
PLoS Biol ; 22(2): e3002502, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38421949

RESUMO

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints.


Assuntos
Revisão por Pares , Pesquisadores , Humanos , Movimento (Física)
3.
Cancer Discov ; 14(2): 240-257, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-37916956

RESUMO

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.


Assuntos
Neoplasias da Mama , Hiperinsulinismo , Humanos , Feminino , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Microscopia Crioeletrônica , Neoplasias da Mama/tratamento farmacológico , Classe I de Fosfatidilinositol 3-Quinases/genética , Hiperinsulinismo/tratamento farmacológico , Hiperinsulinismo/genética , DNA
4.
bioRxiv ; 2023 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-37965202

RESUMO

In hereditary papillary renal cell carcinoma (HPRCC), the MET receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations recently identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, further enhanced by HGF stimulation: His1086Leu, Ile1102Thr, Leu1130Ser, and Cis1125Gly. Similar to the variant resulting in MET exon14 skipping, the two N-lobe MET variants His1086Leu, Ile1102Thr further characterized were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation displayed also transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET TKIs opens the way for use of targeted therapies for patients harboring the corresponding mutations.

5.
Science ; 381(6662): 1092-1098, 2023 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-37676935

RESUMO

Dietary fiber improves metabolic health, but host-encoded mechanisms for digesting fibrous polysaccharides are unclear. In this work, we describe a mammalian adaptation to dietary chitin that is coordinated by gastric innate immune activation and acidic mammalian chitinase (AMCase). Chitin consumption causes gastric distension and cytokine production by stomach tuft cells and group 2 innate lymphoid cells (ILC2s) in mice, which drives the expansion of AMCase-expressing zymogenic chief cells that facilitate chitin digestion. Although chitin influences gut microbial composition, ILC2-mediated tissue adaptation and gastrointestinal responses are preserved in germ-free mice. In the absence of AMCase, sustained chitin intake leads to heightened basal type 2 immunity, reduced adiposity, and resistance to obesity. These data define an endogenous metabolic circuit that enables nutrient extraction from an insoluble dietary constituent by enhancing digestive function.


Assuntos
Adaptação Fisiológica , Quitina , Quitinases , Fibras na Dieta , Obesidade , Estômago , Animais , Camundongos , Quitina/metabolismo , Imunidade Inata , Linfócitos/enzimologia , Linfócitos/imunologia , Obesidade/imunologia , Estômago/imunologia , Adaptação Fisiológica/imunologia , Quitinases/metabolismo , Digestão/imunologia
6.
Nat Chem ; 15(11): 1549-1558, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37723259

RESUMO

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.


Assuntos
Proteínas , Cristalografia por Raios X , Modelos Moleculares , Temperatura , Proteínas/química , Conformação Molecular , Conformação Proteica
7.
Methods Enzymol ; 688: 223-254, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37748828

RESUMO

Conformational ensembles underlie all protein functions. Thus, acquiring atomic-level ensemble models that accurately represent conformational heterogeneity is vital to deepen our understanding of how proteins work. Modeling ensemble information from X-ray diffraction data has been challenging, as traditional cryo-crystallography restricts conformational variability while minimizing radiation damage. Recent advances have enabled the collection of high quality diffraction data at ambient temperatures, revealing innate conformational heterogeneity and temperature-driven changes. Here, we used diffraction datasets for Proteinase K collected at temperatures ranging from 313 to 363 K to provide a tutorial for the refinement of multiconformer ensemble models. Integrating automated sampling and refinement tools with manual adjustments, we obtained multiconformer models that describe alternative backbone and sidechain conformations, their relative occupancies, and interconnections between conformers. Our models revealed extensive and diverse conformational changes across temperature, including increased bound peptide ligand occupancies, different Ca2+ binding site configurations and altered rotameric distributions. These insights emphasize the value and need for multiconformer model refinement to extract ensemble information from diffraction data and to understand ensemble-function relationships.


Assuntos
Difração de Raios X , Temperatura , Cristalografia , Sítios de Ligação , Domínios Proteicos
8.
J Am Chem Soc ; 145(32): 17632-17642, 2023 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-37535945

RESUMO

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with ß-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.


Assuntos
Fluordesoxiglucose F18 , Trealose , Humanos , Celobiose , Staphylococcus aureus , Tomografia por Emissão de Pósitrons/métodos , Bactérias
9.
PLoS Pathog ; 19(8): e1011614, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37651466

RESUMO

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus , SARS-CoV-2 , Replicação Viral , Animais , Humanos , Camundongos , Alanina , Antivirais , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo
10.
bioRxiv ; 2023 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-37577651

RESUMO

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of MET intracellular kinase domain in two fusion protein backgrounds: wild type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase αC helix, pointing to differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a ß5 motif that acts as a structural pivot for kinase domain activation in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

11.
bioRxiv ; 2023 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-37398339

RESUMO

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

12.
bioRxiv ; 2023 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-37425870

RESUMO

With the advent of AlphaFold, protein structure prediction has attained remarkable accuracy. These achievements resulted from a focus on single static structures. The next frontier in this field involves enhancing our ability to model conformational ensembles, not just the ground states of proteins. Notably, deposited structures result from interpretation of density maps, which are derived from either X-ray crystallography or cryogenic electron microscopy (cryo-EM). These maps represent ensemble averages, reflecting molecules in multiple conformations. Here, we present the latest developments in qFit, an automated computational approach to model protein conformational heterogeneity into density maps. We present algorithmic advancements to qFit, validated by improved Rfree and geometry metrics across a broad and diverse set of proteins. Automated multiconformer modeling holds significant promise for interpreting experimental structural biology data and for generating novel hypotheses linking macromolecular conformational dynamics to function.

13.
bioRxiv ; 2023 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-37293043

RESUMO

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach, that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[ 18 F]-fluoro-D-glucose ([ 18 F]FDG), the most common tracer used in clinical imaging, to form [ 18 F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [ 18 F]FDG was reacted with ß-D-glucose-1-phosphate in the presence of maltose phosphorylase, both the α-1,4 and α-1,3-linked products 2-deoxy-[ 18 F]-fluoro-maltose ([ 18 F]FDM) and 2-deoxy-2-[ 18 F]-fluoro-sakebiose ([ 18 F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[ 18 F]fluoro-trehalose ([ 18 F]FDT), 2-deoxy-2-[ 18 F]fluoro-laminaribiose ([ 18 F]FDL), and 2-deoxy-2-[ 18 F]fluoro-cellobiose ([ 18 F]FDC). We subsequently tested [ 18 F]FDM and [ 18 F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. The lead sakebiose-derived tracer [ 18 F]FSK was stable in human serum and showed high uptake in preclinical models of myositis and vertebral discitis-osteomyelitis. Both the synthetic ease, and high sensitivity of [ 18 F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [ 18 F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

14.
bioRxiv ; 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37131711

RESUMO

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wildtype. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels compared to the wildtype virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection and showed no signs of lung pathology. Our data validate the SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a promising target to develop antivirals.

15.
bioRxiv ; 2023 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-37205593

RESUMO

Conformational ensembles underlie all protein functions. Thus, acquiring atomic-level ensemble models that accurately represent conformational heterogeneity is vital to deepen our understanding of how proteins work. Modeling ensemble information from X-ray diffraction data has been challenging, as traditional cryo-crystallography restricts conformational variability while minimizing radiation damage. Recent advances have enabled the collection of high quality diffraction data at ambient temperatures, revealing innate conformational heterogeneity and temperature-driven changes. Here, we used diffraction datasets for Proteinase K collected at temperatures ranging from 313 to 363K to provide a tutorial for the refinement of multiconformer ensemble models. Integrating automated sampling and refinement tools with manual adjustments, we obtained multiconformer models that describe alternative backbone and sidechain conformations, their relative occupancies, and interconnections between conformers. Our models revealed extensive and diverse conformational changes across temperature, including increased bound peptide ligand occupancies, different Ca2+ binding site configurations and altered rotameric distributions. These insights emphasize the value and need for multiconformer model refinement to extract ensemble information from diffraction data and to understand ensemble-function relationships.

16.
PLoS Pathog ; 19(3): e1011146, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36862759

RESUMO

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs in the USA are infected with A. caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A. caninum in greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 ß-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A. caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 ß-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistant A. caninum isolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans ß-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A. caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms.


Assuntos
Ancylostoma , Anti-Helmínticos , Animais , Cães , Ancylostoma/genética , Ancylostomatoidea , Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Caenorhabditis elegans , Resistência a Medicamentos/genética , Mutação , Tubulina (Proteína)/genética
17.
Cell ; 186(4): 864-876.e21, 2023 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-36750095

RESUMO

A fundamental strategy of eukaryotic antiviral immunity involves the cGAS enzyme, which synthesizes 2',3'-cGAMP and activates the effector STING. Diverse bacteria contain cGAS-like enzymes that produce cyclic oligonucleotides and induce anti-phage activity, known as CBASS. However, this activity has only been demonstrated through heterologous expression. Whether bacteria harboring CBASS antagonize and co-evolve with phages is unknown. Here, we identified an endogenous cGAS-like enzyme in Pseudomonas aeruginosa that generates 3',3'-cGAMP during phage infection, signals to a phospholipase effector, and limits phage replication. In response, phages express an anti-CBASS protein ("Acb2") that forms a hexamer with three 3',3'-cGAMP molecules and reduces phospholipase activity. Acb2 also binds to molecules produced by other bacterial cGAS-like enzymes (3',3'-cUU/UA/UG/AA) and mammalian cGAS (2',3'-cGAMP), suggesting broad inhibition of cGAS-based immunity. Upon Acb2 deletion, CBASS blocks lytic phage replication and lysogenic induction, but rare phages evade CBASS through major capsid gene mutations. Altogether, we demonstrate endogenous CBASS anti-phage function and strategies of CBASS inhibition and evasion.


Assuntos
Bactérias , Bacteriófagos , Animais , Bactérias/imunologia , Bactérias/virologia , Bacteriófagos/fisiologia , Imunidade , Nucleotidiltransferases/metabolismo
18.
Genome Biol ; 24(1): 36, 2023 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-36829241

RESUMO

Insertions and deletions (indels) enable evolution and cause disease. Due to technical challenges, indels are left out of most mutational scans, limiting our understanding of them in disease, biology, and evolution. We develop a low cost and bias method, DIMPLE, for systematically generating deletions, insertions, and missense mutations in genes, which we test on a range of targets, including Kir2.1. We use DIMPLE to study how indels impact potassium channel structure, disease, and evolution. We find deletions are most disruptive overall, beta sheets are most sensitive to indels, and flexible loops are sensitive to deletions yet tolerate insertions.


Assuntos
Mutação de Sentido Incorreto , Proteínas , Mutação , Proteínas/genética , Mutação INDEL , Biologia
19.
Proc Natl Acad Sci U S A ; 120(2): e2212931120, 2023 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-36598939

RESUMO

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Cristalografia , Pandemias , Ligantes , Simulação de Acoplamento Molecular , Inibidores de Proteases/farmacologia , Antivirais/farmacologia , Antivirais/química
20.
Acta Crystallogr D Struct Biol ; 79(Pt 1): 50-65, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36601807

RESUMO

It is investigated whether molecular-dynamics (MD) simulations can be used to enhance macromolecular crystallography (MX) studies. Historically, protein crystal structures have been described using a single set of atomic coordinates. Because conformational variation is important for protein function, researchers now often build models that contain multiple structures. Methods for building such models can fail, however, in regions where the crystallographic density is difficult to interpret, for example at the protein-solvent interface. To address this limitation, a set of MD-MX methods that combine MD simulations of protein crystals with conventional modeling and refinement tools have been developed. In an application to a cyclic adenosine monophosphate-dependent protein kinase at room temperature, the procedure improved the interpretation of ambiguous density, yielding an alternative water model and a revised protein model including multiple conformations. The revised model provides mechanistic insights into the catalytic and regulatory interactions of the enzyme. The same methods may be used in other MX studies to seek mechanistic insights.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Conformação Proteica , Proteínas/química , Solventes/química , Cristalografia por Raios X
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