Small Molecule Inhibitors of Activation-Induced Deaminase Decrease Class Switch Recombination in B Cells.

Activation-induced deaminase (AID) not only mutates DNA within the immunoglobulin loci to generate antibody diversity, but it also promotes development of B cell lymphomas. To tame this mutagen, we performed a quantitative high-throughput screen of over 90 000 compounds to see if AID activity could be mitigated. The enzymatic activity was assessed in biochemical assays to detect cytosine deamination and in cellular assays to measure class switch recombination. Three compounds showed promise via inhibition of switching in a transformed B cell line and in murine splenic B cells. These compounds have similar chemical structures, which suggests a shared mechanism of action. Importantly, the inhibitors blocked AID, but not a related cytosine DNA deaminase, APOBEC3B. We further determined that AID was continually expressed for several days after B cell activation to induce switching. This first report of small molecules that inhibit AID can be used to gain regulatory control over base editors.

Synchronous Monitoring of Cement Hydration and Polymer Film Formation Using 1H-Time-Domain-NMR with T 2 Time-Weighted T 1 Time Evaluation: A Nondestructive Practicable Benchtop Method.

The interactions between latex and cement are still not completely understood. In this work, we would like to address the temporal changes in cement hardening and latex film formation. For this reason, the hydration process and the film formation were simultaneously monitored. This scientific issue is even more challenging as a nondestructive quantitative analysis of the film formation process is not available yet. Here, we report on simultaneous monitoring of the latex film formation and the phase development in cementitious systems via 1H-time-domain-NMR for the first time. The obtained results were validated using classical analytical methods, such as in situ X-ray diffraction, X-ray fluorescence (Rietveld analysis), and confocal laser scanning microscopy.

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

Phase Solubility Changes during Hydration of Monocalciumaluminate and Calcite-The Influence of Alkali Accumulation.

The reaction of CA (monocalcium aluminate) with calcite was closely monitored with regard to phase development, pore water ion content and heat flow. Calcite acts as filler and reactant, finally leading to thermodynamically stable products after hydration at ambient conditions. For better understanding the mechanism taking place, a CA-cement and a commercial calcite mix were compared to a pure CA and pure calcite mix. Both reaction paths were compared. Thermodynamic modeling with PhreeqC gave insight about factors that can influence the course of the hydration reaction. Alkali ions in pore solution of the CA-cement relocate solubility curves of hydration products. Taking into account as many of the alkaline ions as possible, resulted in the closest representation of the measured phase content, confirming thermodynamic modeling. The high dynamics that develop during reaction could only be addressed if a concentration of alkalis in the pore solution at later points in time was respected, thus leading to a shift of solubility curves over time. This was not observed with the pure CA in absence of alkalis.

Investigation of the Incompatibilities of Cement and Superplasticizers and Their Influence on the Rheological Behavior.

The rheological behavior of cement paste and the improvement of its flowability takes center stage in many research projects. An improved flowability can be achieved by the addition of superplasticizers (SP), such as polycarboxylate ethers (PCE). In order to be able to use these PCEs effectively and in a variety of ways and to make them resistant to changes in the environment, it is crucial to understand the influence of SPs on cement hydration. For that reason, the topic of this paper was the incompatibility of a specific SP and an ordinary Portland cement (OPC). The incompatible behavior was analyzed using rheological tests, such as the spread flow test and penetration test, and the behavior was compared by means of an ultrasound technique and explained by the phase content measured by in-situ X-ray diffraction (XRD) the heat evolution measured by calorimetry, and scanning electron microscope (SEM) images. We showed that the addition of the SP in a high dosage led to a prevention of the passivation of the most reactive and aluminum-containing clinker phases, aluminate and brownmillerite. This induced the aluminate reaction to take place in the initial period and led to an immediate stiffening of the cement paste and, therefore, to the complete loss of workability. The results showed that in addition to the ettringite, which began to form directly after water addition, hemicarbonate precipitated. The fast stiffening of the paste could be prevented by delayed addition of the SP or by additional gypsum. This fast stiffening was not desirable for SPs, but in other fields, for example, 3D printing, this undesirable interaction could be used to improve the properties of printable mortar.

Relating Ettringite Formation and Rheological Changes during the Initial Cement Hydration: A Comparative Study Applying XRD Analysis, Rheological Measurements and Modeling.

In order to gain a deeper understanding of the rheological development of hydrating ordinary Portland cement (OPC) pastes at initial state, and to better understand their underlying processes, quantitative X-ray diffraction (XRD) analysis and rheological measurements were conducted and their results combined. The time-dependent relation between phase development and flow behavior of cement paste was investigated at two different temperatures (20 and 30 °C), over a period of two hours. Regarding the phase development during hydration, ettringite precipitation was identified as the dominant reaction in the first two hours. For both temperatures, the increasing ettringite content turned out to correlate very well with the loss of workability of the reacting cement paste. An exponential relationship between ettringite growth and flow behavior was observed that could be explained by applying the Krieger-Dougherty equation, which describes the influence of solid fraction on the viscosity of a suspension.

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics.

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC quality control is compromised in aging mitotic cells. Our images of single yeast cells during aging, show that the abundance of several NPC components and NPC assembly factors decreases. Additionally, the single-cell life histories reveal that cells that better maintain those components are longer lived. The presence of herniations at the nuclear envelope of aged cells suggests that misassembled NPCs are accumulated in aged cells. Aged cells show decreased dynamics of transcription factor shuttling and increased nuclear compartmentalization. These functional changes are likely caused by the presence of misassembled NPCs, as we find that two NPC assembly mutants show similar transport phenotypes as aged cells. We conclude that NPC interphase assembly is a major challenge for aging mitotic cells.

KDM5B Promotes Drug Resistance by Regulating Melanoma-Propagating Cell Subpopulations.

Tumor heterogeneity is a major challenge for cancer treatment, especially due to the presence of various subpopulations with stem cell or progenitor cell properties. In mouse melanomas, both CD34+p75- (CD34+) and CD34-p75- (CD34-) tumor subpopulations were characterized as melanoma-propagating cells (MPC) that exhibit some of those key features. However, these two subpopulations differ from each other in tumorigenic potential, ability to recapitulate heterogeneity, and chemoresistance. In this study, we demonstrate that CD34+ and CD34- subpopulations carrying the BRAFV600E mutation confer differential sensitivity to targeted BRAF inhibition. Through elevated KDM5B expression, melanoma cells shift toward a more drug-tolerant, CD34- state upon exposure to BRAF inhibitor or combined BRAF inhibitor and MEK inhibitor treatment. KDM5B loss or inhibition shifts melanoma cells to the more BRAF inhibitor-sensitive CD34+ state. These results support that KDM5B is a critical epigenetic regulator that governs the transition of key MPC subpopulations with distinct drug sensitivity. This study also emphasizes the importance of continuing to advance our understanding of intratumor heterogeneity and ultimately develop novel therapeutics by altering the heterogeneous characteristics of melanoma.

Structure-Based Engineering of Irreversible Inhibitors against Histone Lysine Demethylase KDM5A.

The active sites of hundreds of human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a noncatalytic cysteine (Cys481 in KDM5A) near the active sites of KDM5 histone H3 lysine 4 demethylases, which is absent in other histone demethylase families, that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely, 2-((3-aminophenyl)(2-(piperidin-1-yl)ethoxy)methyl)thieno[3,2- b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDM5A and determined the cocrystal structures of the catalytic domain of KDM5A in complex with N70 and N71. Whereas the noncovalent inhibitor N70 displayed αKG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.

KDM5 histone demethylases repress immune response via suppression of STING.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) stimulator of interferon genes (STING) senses pathogen-derived or abnormal self-DNA in the cytosol and triggers an innate immune defense against microbial infection and cancer. STING agonists induce both innate and adaptive immune responses and are a new class of cancer immunotherapy agents tested in multiple clinical trials. However, STING is commonly silenced in cancer cells via unclear mechanisms, limiting the application of these agonists. Here, we report that the expression of STING is epigenetically suppressed by the histone H3K4 lysine demethylases KDM5B and KDM5C and is activated by the opposing H3K4 methyltransferases. The induction of STING expression by KDM5 blockade triggered a robust interferon response in a cytosolic DNA-dependent manner in breast cancer cells. This response resulted in resistance to infection by DNA and RNA viruses. In human tumors, KDM5B expression is inversely associated with STING expression in multiple cancer types, with the level of intratumoral CD8+ T cells, and with patient survival in cancers with a high level of cytosolic DNA, such as human papilloma virus (HPV)-positive head and neck cancer. These results demonstrate a novel epigenetic regulatory pathway of immune response and suggest that KDM5 demethylases are potential targets for antipathogen treatment and anticancer immunotherapy.

Insights into the Action of Inhibitor Enantiomers against Histone Lysine Demethylase 5A.

Isomers of chiral drugs can exhibit marked differences in biological activities. We studied the binding and inhibitory activities of 12 compounds against KDM5A. Among them are two pairs of enantiomers representing two distinct inhibitor chemotypes, namely, ( R)- and ( S)-2-((2-chlorophenyl)(2-(piperidin-1-yl)ethoxy)methyl)-1 H-pyrrolo[3,2- b]pyridine-7-carboxylic acid (compounds N51 and N52) and ( R) - and ( S) -N-(1-(3-isopropyl-1 H-pyrazole-5-carbonyl)pyrrolidin-3-yl)cyclopropanecarboxamide (compounds N54 and N55). In vitro, the S enantiomer of the N51/N52 pair (N52) and the R enantiomer of the N54/N55 pair (N54) exhibited about 4- to 5-fold greater binding affinity. The more potent enzyme inhibition of KDM5A by the R-isoform for the cell-permeable N54/N55 pair translated to differences in growth inhibitory activity. We determined structures of the KDM5A catalytic domain in complex with all 12 inhibitors, which revealed the interactions (or lack thereof) responsible for the differences in binding affinity. These results provide insights to guide improvements in binding potency and avenues for development of cell permeable inhibitors of the KDM5 family.

Synthesis and Sulfur Electrophilicity of the Nuphar Thiaspirane Pharmacophore.

We describe a general method to synthesize the iminium tetrahydrothiophene embedded in the dimeric Nuphar alkaloids. In contrast to prior studies, the sulfur atom of the thiaspirane pharmacophore is shown to be electrophilic. This α-thioether reacts with thiophenol or glutathione at ambient temperature to cleave the C-S bond and form a disulfide. Rates of conversion are proportional to the corresponding ammonium ion pK a and exhibit half-lives less than 5 h at a 5 mM concentration of thiol. A simple thiophane analogue of the Nuphar dimers causes apoptosis at single-digit micromolar concentration and labels reactive cysteines at similar levels as the unsaturated iminium "warhead". Our experiments combined with prior observations suggest the sulfur of the Nuphar dimers can react as an electrophile in cellular environments and that sulfur-triggered retrodimerization can occur in the cell.

Structural Basis for KDM5A Histone Lysine Demethylase Inhibition by Diverse Compounds.

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases removes methyl groups from methylated lysine 4 of histone H3. Accumulating evidence supports a role for KDM5 family members as oncogenic drivers. We compare the in vitro inhibitory properties and binding affinity of ten diverse compounds with all four family members, and present the crystal structures of the KDM5A-linked Jumonji domain in complex with eight of these inhibitors in the presence of Mn(II). All eight inhibitors structurally examined occupy the binding site of α-ketoglutarate, but differ in their specific binding interactions, including the number of ligands involved in metal coordination. We also observed inhibitor-induced conformational changes in KDM5A, particularly those residues involved in the binding of α-ketoglutarate, the anticipated peptide substrate, and intramolecular interactions. We discuss how particular chemical moieties contribute to inhibitor potency and suggest strategies that might be utilized in the successful design of selective and potent epigenetic inhibitors.

Three-dimensional Cervical Movement Characteristics in Healthy Subjects and Subgroups of Chronic Neck Pain Patients Based on Their Pain Location.

STUDY DESIGN: A cross-sectional observational study of three-dimensional (3D) cervical kinematics in 41 chronic neck pain (CNPs) patients and 156 asymptomatic controls. OBJECTIVE: The objective was to investigate 3D cervical kinematics by analyzing and comparing quantitative and qualitative parameters in healthy subjects and CNPs. Furthermore, subgroups were formed to explore the influence of pain-location on cervical kinematics. The possible correlation of kinematic parameters with the degree of functional disability was examined as well. SUMMARY OF BACKGROUND DATA: In patients with chronic neck pain, a clear pathological cause is frequently not identifiable. Therefore, the need to assess neck pain with a broader view than structure or anatomical-based divergences is desirable. METHODS: Movements of the cervical spine were registered using an electromagnetic tracking system. Quantitative and qualitative kinematics were analyzed for active axial rotation, lateral bending, and flexion-extension motion components. RESULTS: During lateral bending, the range of the main motion demonstrated significant higher values (P = 0.001) in the controls (mean: 68.67°â€Š±â€Š15.17°) than patients (mean: 59.28°â€Š±â€Š15.41°). Significant differences were demonstrated between subgroups for several kinematic parameters (P < 0.05). Although differences were predominantly recorded between the "symmetrical" and "asymmetrical" pain group, some parameters also distinguished subgroups from controls. On average, the symmetrical group showed significant less harmonic movement patterns, expressed by qualitative parameters, in comparison with the "asymmetrical" group and controls. Furthermore, the "asymmetrical" group showed significant lower scores on quantitative parameters than the "symmetrical" group and controls. The degree of functional disability correlated moderately with changes in qualitative parameters. CONCLUSION: In this study, chronic neck pain patients with a symmetrical pain pattern showed significant poorer quality of movement, while those with asymmetrical pain showed a significant reduction in quantitative measures. Subgrouping of neck patients based on pain location may be of help for further research and clinics. LEVEL OF EVIDENCE: 4.

Synthesis of medicinally relevant terpenes: reducing the cost and time of drug discovery.

Terpenoids constitute a significant fraction of molecules produced by living organisms that have found use in medicine and other industries. Problems associated with their procurement and adaptation for human use can be solved using chemical synthesis, which is an increasingly economical option in the modern era of chemistry. This article documents, by way of individual case studies, strategies for reducing the time and cost of terpene synthesis for drug discovery. A major trend evident in recent syntheses is that complex terpenes are increasingly realistic starting points for both medicinal chemistry campaigns and large-scale syntheses, at least in the context of the academic laboratory, and this trend will likely penetrate the commercial sector in the near future.

Synthesis of (-)-neothiobinupharidine.

An eight step, asymmetric synthesis of a dimeric thiaspirane nuphar alkaloid from 3-methyl-2-cyclo-pentenone is reported. The brevity of the route relies on a useful procedure for tandem reductive allylation of cyclopentenones, as well as the minimization of redox manipulations and other functional group interconversions. The distribution of products that arise from spontaneous dimerization points to a more complex biosynthesis.

A stereoselective hydroamination transform to access polysubstituted indolizidines.

Stereoselective, intramolecular, formal hydroamination of dienamines via directed hydroboration is reported. Four stereocenters are set in the process. Natural and unnatural indolizidine alkaloids can be synthesized from simple unsaturated amines using the title process.

Intramolecular azide-alkyne [3 + 2] cycloaddition: versatile route to new heterocyclic structural scaffolds.

Investigating the relatively unexplored intramolecular version of the azide-alkyne [3 + 2] cycloaddition, the present studies demonstrate the utility of the above reaction in the synthesis of a variety of as yet unreported heterocyclic structural scaffolds. The approach involved initial installation of strategic azide and alkyne moieties on a common structural framework, followed by their intramolecular cycloaddition studies. The pivotal azidoalkyne intermediates were efficiently accessed from a variety of easily available starting materials such as olefins, epoxides, amino acids, amino alcohols, ketones etc. The key reactions for incorporation of the azide functionality into the desired framework involved azidolysis of epoxides, displacement of hydroxy groups with azide nucleophiles, and diazo transfer on amine. Attachment of the desired alkyne functionalities was accomplished by either N-, or, O-alkylation with appropriate propargylic halides. The azidoalkynes thus prepared underwent smooth intramolecular cycloaddition, resulting in a variety of novel triazolooxazine and triazolopyrazine derivatives. Interestingly, unlike in the intermolecular version, metal catalysis was not necessary for the performance of the above cycloadditions. It is expected that the results from the present studies and its further extension will provide a potentially fertile pathway to a variety of unique chemical entities of structural and biological significance.