Identification of a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) mutant with improved crystallographic properties

In this report, we describe a truncated Deinococcus radiodurans 1-deoxy-D-xylulose-5-phosphate synthase (DXS) protein that retains enzymatic activity, while slowing protein degradation and showing improved crystallization properties. With modern drug-design approaches relying heavily on the elucidation of atomic interactions of potential new drugs with their targets, the need for co-crystal structures with the compounds of interest is high. DXS itself is a promising drug target, as it catalyzes the first reaction in the 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway for the biosynthesis of the universal precursors of terpenes, which are essential secondary metabolites. In contrast to many bacteria and pathogens, which employ the MEP pathway, mammals use the distinct mevalonate-pathway for the biosynthesis of these precursors, which makes all enzymes of the MEP-pathway potential new targets for the development of anti-infectives. However, crystallization of DXS has proven to be challenging: while the first X-ray structures from Escherichia coli and D. radiodurans were solved in 2004, since then only two additions have been made in 2019 that were obtained under anoxic conditions. The presented site of truncation can potentially also be transferred to other homologues, opening up the possibility for the determination of crystal structures from pathogenic species, which until now could not be crystallized. This manuscript also provides a further example that truncation of a variable region of a protein can lead to improved structural data

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 frst 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 frst DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main diference 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 identifed in the enamine structure, using a diferent 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 specifc for MtDXPS through structure-based drug design

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

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage

Qualitative prediction of blood–brain barrier permeability on a large and refined dataset

The prediction of blood–brain barrier permeation is vitally important for the optimization of drugs targeting the central nervous system as well as for avoiding side effects of peripheral drugs. Following a previously proposed model on blood–brain barrier penetration, we calculated the cross-sectional area perpendicular to the amphiphilic axis. We obtained a high correlation between calculated and experimental cross-sectional area (r = 0.898, n = 32). Based on these results, we examined a correlation of the calculated cross-sectional area with blood–brain barrier penetration given by logBB values. We combined various literature data sets to form a large-scale logBB dataset with 362 experimental logBB values. Quantitative models were calculated using bootstrap validated multiple linear regression. Qualitative models were built by a bootstrapped random forest algorithm. Both methods found similar descriptors such as polar surface area, pKa, logP, charges and number of positive ionisable groups to be predictive for logBB. In contrast to our initial assumption, we were not able to obtain models with the cross-sectional area chosen as relevant parameter for both approaches. Comparing those two different techniques, qualitative random forest models are better suited for blood-brain barrier permeability prediction, especially when reducing the number of descriptors and using a large dataset. A random forest prediction system (ntrees = 5) based on only four descriptors yields a validated accuracy of 88%

Improved upper limb function in non-ambulant children with SMA type 2 and 3 during nusinersen treatment: a prospective 3-years SMArtCARE registry study

Background The development and approval of disease modifying treatments have dramatically changed disease progression in patients with spinal muscular atrophy (SMA). Nusinersen was approved in Europe in 2017 for the treatment of SMA patients irrespective of age and disease severity. Most data on therapeutic efficacy are available for the infantile-onset SMA. For patients with SMA type 2 and type 3, there is still a lack of sufficient evidence and long-term experience for nusinersen treatment. Here, we report data from the SMArtCARE registry of non-ambulant children with SMA type 2 and typen 3 under nusinersen treatment with a follow-up period of up to 38 months. Methods SMArtCARE is a disease-specific registry with data on patients with SMA irrespective of age, treatment regime or disease severity. Data are collected during routine patient visits as real-world outcome data. This analysis included all non-ambulant patients with SMA type 2 or 3 below 18 years of age before initiation of treatment. Primary outcomes were changes in motor function evaluated with the Hammersmith Functional Motor Scale Expanded (HFMSE) and the Revised Upper Limb Module (RULM). Results Data from 256 non-ambulant, pediatric patients with SMA were included in the data analysis. Improvements in motor function were more prominent in upper limb: 32.4% of patients experienced clinically meaningful improvements in RULM and 24.6% in HFMSE. 8.6% of patients gained a new motor milestone, whereas no motor milestones were lost. Only 4.3% of patients showed a clinically meaningful worsening in HFMSE and 1.2% in RULM score. Conclusion Our results demonstrate clinically meaningful improvements or stabilization of disease progression in non-ambulant, pediatric patients with SMA under nusinersen treatment. Changes were most evident in upper limb function and were observed continuously over the follow-up period. Our data confirm clinical trial data, while providing longer follow-up, an increased number of treated patients, and a wider range of age and disease severity

Residue analyses for an updated evaluation of the application of hormonal growth promoters in animal fattening

Bei der Anwendung hormonal wirksamer Leistungsförderer in der Tiermast in Form von Implantationspräparaten ist die Einhaltung der "Guten Veterinärpraxis" unabdingbar. Gelangen Implantationsstellen in die Nahrungskette, können ganze Chargen von Fleischprodukten kontaminiert werden. Das höchste Gefährdungspotential geht dabei von Trenbolonacetatpräparaten aus, gefolgt von Östradiol bzw. Östradiolbenzoat, Testosteronpropionat und Progesteron. Eine Evaluierung von Zeranol war diesbezüglich im Rahmen der vorliegenden Untersuchung nicht möglich. Bei Mehrfachdosierung können in peripheren Geweben weder nach Behandlung mit Zeranol noch mit Testosteronpropionat unzulässig hohe Rückstände detektiert werden. Missbräuchliche Anwendung von Trenbolonacetat und Östradiolbenzoat hingegen kann zur Verletzung erlaubter Toleranzgrenzen führen. Eine Überschreitung des MRL für Leber wurde in einem von zwei Tieren nach Applikation von 600 mg und bei zwei Tieren nach Applikation von 2000 mg Trenbolonacetat festgestellt. Die U. S.-Östradiolgrenzwerte wurden in Leber und in Niere schon nach dreifacher Dosis von Synovex-H® überschritten. Die Behandlung von Kälbern mit Synovex-H® und Synovex Plus® führte zu ähnlichen Rückstandsniveaus wie nach Behandlung von Färsen mit Synovex-H® bzw. Finaplix-H®. Mögliche Auswirkungen von Rückständen hormonaler Leistungsförderer in der Nahrung müssen stets im Zusammenhang mit der Gesamtbelastung durch "endocrine disruptors" gesehen werden. Besonderes Augenmerk gilt dabei möglichen irreversiblen Effekten auf die pränatale und präpubertäre Entwicklung. Der plazentale Transfer von Trenbolon, Zeranol und Melengestrolacetat konnte beim Kaninchen anhand von Rückstandsuntersuchungen fötaler Gewebe gezeigt werden, d. h. diese Verbindungen passieren die Plazentaschranke und können so möglicherweise in utero die Organogenese beeinträchtigen.Application of hormonal growth promoting implant preparations in animal fattening has to take place in observance of Good Vetrinary Practice. Implantation sites reaching the food chain can contaminate whole batches of processed meat. The highest potential risk comes from trenbolone acetate preparations, followed by estradiol or estradiol benzoate, testosterone propionate and progesterone. An evaluation of zeranol was not possible within the scope of this study. After multiple application, treatment with zeranol or testosterone propionate does not cause any infringement of threshold levels in peripheral tissues. Misuse of trenbolone acetate and estradiol benzoate, however, may lead to illicit values. Exceeding of the MRL was found in liver in one out of two animals after treatment with 600 mg and in two out of two animals after treatment with 2000 mg trenbolone acetate. U. S. estradiol threshold levels were violated in the liver and in the kidney even after 3-fold dose of Synovex-H®. Treatment of calves with Synovex-H® or Synovex Plus® led to similar residue levels as after Synovex-H® or Finaplix-H® treatment of heifers. Possible effects of residual hormonal growth promoters in food have to be connected with the overall burden of endocrine disruptors. Attention has to be directed to irreversible effects on prenatal and prepubertal development. The placental transfer of trenbolone, zeranol and melengestrol acetate could be shown in rabbit after residue analysis of fetal tissues. These substances pass the placental barrier and could possibly impair organogenesis in utero