Small-Molecule Allosteric Inhibitors of Human Aspartate Transcarbamoylase Suppress Proliferation of Bone Osteosarcoma Epithelial Cells.

Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120 nM) in an in vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10 µM. Evidence for a druggable allosteric pocket in human ATC is provided by both in vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.

Engineering a Plant Polyketide Synthase for the Biosynthesis of Methylated Flavonoids.

Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce various flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of O-methylated flavonoids is currently still highly inefficient. In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperetin production in Escherichia coli from fed O-methylated hydroxycinnamic acids. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from Hordeum vulgare, in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in E. coli toward producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234 V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than reported previously. Our findings will facilitate further engineering of this enzyme toward higher production of methylated flavonoids.

Thiosulfate sulfurtransferase deficiency promotes oxidative distress and aberrant NRF2 function in the brain.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1) was discovered as an enzyme that detoxifies cyanide by conversion to thiocyanate (rhodanide) using thiosulfate as substrate; this rhodanese activity was subsequently identified to be almost exclusively located in mitochondria. More recently, the emphasis regarding its function has shifted to hydrogen sulfide metabolism, antioxidant defense, and mitochondrial function in the context of protective biological processes against oxidative distress. While TST has been described to play an important role in liver and colon, its function in the brain remains obscure. In the present study, we therefore sought to address its potential involvement in maintaining cerebral redox balance in a murine model of global TST deficiency (Tst-/- mice), primarily focusing on characterizing the biochemical phenotype of TST loss in relation to neuronal activity and sensitivity to oxidative stress under basal conditions. Here, we show that TST deficiency is associated with a perturbation of the reactive species interactome in the brain cortex secondary to altered ROS and RSS (specifically, polysulfide) generation as well as mitochondrial OXPHOS remodeling. These changes were accompanied by aberrant Nrf2-Keap1 expression and thiol-dependent antioxidant function. Upon challenging mice with the redox-active herbicide paraquat (25 mg/kg i.p. for 24 h), Tst-/- mice displayed a lower antioxidant capacity compared to wildtype controls (C57BL/6J mice). These results provide a first glimpse into the molecular and metabolic changes of TST deficiency in the brain and suggest that pathophysiological conditions associated with aberrant TST expression and/or activity renders neurons more susceptible to oxidative stress-related malfunction.

Correction: Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment.

[This corrects the article DOI: 10.1371/journal.pone.0196254.].

Identification and characterization of a small molecule that activates thiosulfate sulfurtransferase and stimulates mitochondrial respiration.

The enzyme Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), is a positive genetic predictor of diabetes type 2 and obesity. As increased TST activity protects against the development of diabetic symptoms in mice, an activating compound for TST may provide therapeutic benefits in diabetes and obesity. We identified a small molecule activator of human TST through screening of an inhouse small molecule library. Kinetic studies in vitro suggest that two distinct isomers of the compound are required for full activation as well as an allosteric mode of activation. Additionally, we studied the effect of TST protein and the activator on TST activity through mitochondrial respiration. Molecular docking and molecular dynamics (MD) approaches supports an allosteric site for the binding of the activator, which is supported by the lack of activation in the Escherichia coli. mercaptopyruvate sulfurtransferase. Finally, we show that increasing TST activity in isolated mitochondria increases mitochondrial oxygen consumption.

A comprehensive overview of the heterogeneity of EGFR exon 20 variants in NSCLC and (pre)clinical activity to currently available treatments.

Activating EGFR mutations are commonly observed in non-small cell lung cancer (NSCLC). About 4-10 % of all activating epidermal growth factor receptor (EGFR) mutations are heterogenous in-frame deletion and/or insertion mutations clustering within exon 20 (EGFRex20+). NSCLC patients with EGFRex20+ mutations are treated as a single disease entity, irrespective of the type and location of the mutation. Here, we provide a comprehensive assessment of the literature reporting both in vitro and clinical drug sensitivity across different EGFRex20+ mutations. The activating A763_Y764insFQEA mutation has a better tumor response in comparison with mutations in the near- and far regions directly following the C-helix and should therefore be treated differently. For other EGFRex20+ mutations marked differences in treatment responses have been reported indicating the need for a classification beyond the exon-based classification. A further classification can be achieved using a structure-function modeling approach and experimental data using patient-derived cell lines. The detailed overview of TKI responses for each EGFRex20+ mutation can assist treating physicians to select the most optimal drug for individual NSCLC patients.

Inhibitors of Aspartate Transcarbamoylase Inhibit Mycobacterium tuberculosis Growth.

Aspartate transcarbamoylase (ATCase) plays a key role in the second step of de novo pyrimidine biosynthesis in eukaryotes and has been proposed to be a target to suppress cell proliferation in E. coli, human cells and the malarial parasite. We hypothesized that a library of ATCase inhibitors developed for malarial ATCase (PfATCase) may also contain inhibitors of the tubercular ATCase and provide a similar inhibition of cellular proliferation. Of the 70 compounds screened, 10 showed single-digit micromolar inhibition in an in vitro activity assay and were tested for their effect on M. tuberculosis cell growth in culture. The most promising compound demonstrated a MIC90 of 4 µM. A model of MtbATCase was generated using the experimental coordinates of PfATCase. In silico docking experiments showed this compound can occupy a similar allosteric pocket on MtbATCase to that seen on PfATCase, explaining the observed species selectivity seen for this compound series.

Structural Characterization and Extended Substrate Scope Analysis of Two Mg2+ -Dependent O-Methyltransferases from Bacteria.

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized the enzymatic properties and substrate scope of two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids, and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether, and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering.

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.

Discovery of Small-Molecule Allosteric Inhibitors of PfATC as Antimalarials.

The discovery and development of new drugs against malaria remain urgent. Aspartate transcarbamoylase (ATC) has been suggested to be a promising target for antimalarial drug development. Here, we describe a series of small-molecule inhibitors of P. falciparum ATC with low nanomolar binding affinities that selectively bind to a previously unreported allosteric pocket, thereby inhibiting ATC activation. We demonstrate that the buried allosteric pocket is located close to the traditional ATC active site and that reported compounds maintain the active site of PfATC in its low substrate affinity/low activity conformation. These compounds inhibit parasite growth in blood stage cultures at single digit micromolar concentrations, whereas limited effects were seen against human normal lymphocytes. To our knowledge, this series represent the first PfATC-specific allosteric inhibitors.

Thiosulfate sulfurtransferase prevents hyperglycemic damage to the zebrafish pronephros in an experimental model for diabetes.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate ß-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.

First crystal structures of 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) from Mycobacterium tuberculosis indicate a distinct mechanism of intermediate stabilization.

The development of drug resistance by Mycobacterium tuberculosis and other pathogenic bacteria emphasizes the need for new antibiotics. Unlike animals, most bacteria synthesize isoprenoid precursors through the MEP pathway. 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first reaction of the MEP pathway and is an attractive target for the development of new antibiotics. We report here the successful use of a loop truncation to crystallize and solve the first DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main difference found to other DXPS structures is in the active site where a highly coordinated water was found, showing a new mechanism for the enamine-intermediate stabilization. Unlike other DXPS structures, a "fork-like" motif could be identified in the enamine structure, using a different residue for the interaction with the cofactor, potentially leading to a decrease in the stability of the intermediate. In addition, electron density suggesting a phosphate group could be found close to the active site, provides new evidence for the D-GAP binding site. These results provide the opportunity to improve or develop new inhibitors specific for MtDXPS through structure-based drug design.

Feasibility of Follow-Up Studies and Reclassification in Spinocerebellar Ataxia Gene Variants of Unknown Significance.

Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative disorders with autosomal dominant inheritance. Genetic testing for SCA leads to diagnosis, prognosis and risk assessment for patients and their family members. While advances in sequencing and computing technologies have provided researchers with a rapid expansion in the genetic test content that can be used to unravel the genetic causes that underlie diseases, the large number of variants with unknown significance (VUSes) detected represent challenges. To minimize the proportion of VUSes, follow-up studies are needed to aid in their reclassification as either (likely) pathogenic or (likely) benign variants. In this study, we addressed the challenge of prioritizing VUSes for follow-up using (a combination of) variant segregation studies, 3D protein modeling, in vitro splicing assays and functional assays. Of the 39 VUSes prioritized for further analysis, 13 were eligible for follow up. We were able to reclassify 4 of these VUSes to LP, increasing the molecular diagnostic yield by 1.1%. Reclassification of VUSes remains difficult due to limited possibilities for performing variant segregation studies in the classification process and the limited availability of routine functional tests.

Novel Highlight in Malarial Drug Discovery: Aspartate Transcarbamoylase.

Malaria remains one of the most prominent and dangerous tropical diseases. While artemisinin and analogs have been used as first-line drugs for the past decades, due to the high mutational rate and rapid adaptation to the environment of the parasite, it remains urgent to develop new antimalarials. The pyrimidine biosynthesis pathway plays an important role in cell growth and proliferation. Unlike human host cells, the malarial parasite lacks a functional pyrimidine salvage pathway, meaning that RNA and DNA synthesis is highly dependent on the de novo synthesis pathway. Thus, direct or indirect blockage of the pyrimidine biosynthesis pathway can be lethal to the parasite. Aspartate transcarbamoylase (ATCase), catalyzes the second step of the pyrimidine biosynthesis pathway, the condensation of L-aspartate and carbamoyl phosphate to form N-carbamoyl aspartate and inorganic phosphate, and has been demonstrated to be a promising target both for anti-malaria and anti-cancer drug development. This is highlighted by the discovery that at least one of the targets of Torin2 - a potent, yet unselective, antimalarial - is the activity of the parasite transcarbamoylase. Additionally, the recent discovery of an allosteric pocket of the human homology raises the intriguing possibility of species selective ATCase inhibitors. We recently exploited the available crystal structures of the malarial aspartate transcarbamoylase to perform a fragment-based screening to identify hits. In this review, we summarize studies on the structure of Plasmodium falciparum ATCase by focusing on an allosteric pocket that supports the catalytic mechanisms.

Actionability of on-target ALK Resistance Mutations in Patients With Non-Small Cell Lung Cancer: Local Experience and Review of the Literature.

INTRODUCTION: Non-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) gene fusions respond well to ALK inhibitors but commonly develop on-target resistance mutations. The objective of this study is to collect clinical evidence for subsequent treatment with ALK inhibitors. PATIENTS AND METHODS: Local experience with on-target ALK resistance mutations and review of the literature identified 387 patients with ALK inhibitor resistance mutations. Clinical benefit of mutation-inhibitor combinations was assessed based on reported response, progression-free survival and duration of treatment. Furthermore, this clinical evidence was compared to previously reported in vitro sensitivity of mutations to the inhibitors. RESULTS: Of the pooled population of 387 patients in this analysis, 239 (62%) received at least 1 additional line of ALK inhibition after developing on-target resistance to ALK inhibitor therapy. Clinical benefit was reported for 177 (68%) patients, but differed for each mutation-inhibitor combination. Agreement between in vitro predicted sensitivity of 6 published models and observed clinical benefit ranged from 69% to 89%. The observed clinical evidence for highest probability of response in the context of specific on-target ALK inhibitor resistance mutations is presented. CONCLUSION: Molecular diagnostics performed on tissue samples that are refractive to ALK inhibitor therapy can reveal new options for targeted therapy for NSCLC patients. Our comprehensive overview of clinical evidence of drug actionability of ALK on-target resistance mechanisms may serve as a practical guide to select the most optimal drug for individual patients.

Synthetic Peptides That Antagonize the Angiotensin-Converting Enzyme-2 (ACE-2) Interaction with SARS-CoV-2 Receptor Binding Spike Protein.

The SARS-CoV-2 viral spike protein S receptor-binding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein-protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, ß3, and ß4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.

Lipoic Acid Metabolism as a Potential Chemotherapeutic Target Against Plasmodium falciparum and Staphylococcus aureus.

Lipoic acid (LA) is an organic compound that plays a key role in cellular metabolism. It participates in a posttranslational modification (PTM) named lipoylation, an event that is highly conserved and that occurs in multimeric metabolic enzymes of very distinct microorganisms such as Plasmodium sp. and Staphylococcus aureus, including pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KDH). In this mini review, we revisit the recent literature regarding LA metabolism in Plasmodium sp. and Staphylococcus aureus, by covering the lipoate ligase proteins in both microorganisms, the role of lipoate ligase proteins and insights for possible inhibitors of lipoate ligases.

A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity.

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site.

Combining High-Throughput Synthesis and High-Throughput Protein Crystallography for Accelerated Hit Identification.

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and -esters from diverse building blocks suitable for mmol scale synthesis on 96-well format and on a high-throughput nanoscale format in a highly automated fashion. High-throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID-19 causing agent, SARS-CoV-2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects.

Nanoscale, automated, high throughput synthesis and screening for the accelerated discovery of protein modifiers.

Hit finding in early drug discovery is often based on high throughput screening (HTS) of existing and historical compound libraries, which can limit chemical diversity, is time-consuming, very costly, and environmentally not sustainable. On-the-fly compound synthesis and in situ screening in a highly miniaturized and automated format has the potential to greatly reduce the medicinal chemistry environmental footprint. Here, we used acoustic dispensing technology to synthesize a library in a 1536 well format based on the Groebcke-Blackburn-Bienaymé reaction (GBB-3CR) on a nanomole scale. The unpurified library was screened by differential scanning fluorimetry (DSF) and cross-validated using microscale thermophoresis (MST) against the oncogenic protein-protein interaction menin-MLL. Several GBB reaction products were found as µM menin binder, and the structural basis of the interactions with menin was elucidated by co-crystal structure analysis. Miniaturization and automation of the organic synthesis and screening process can lead to an acceleration in the early drug discovery process, which is an alternative to classical HTS and a step towards the paradigm of continuous manufacturing.