A state of independents: rationalising the high <em>Z</em>\u27 crystal structures of shikimate esters

Zˈ is a parameter used to denote the number of symmetry-independent molecules in the asymmetric unit of a crystal structure. High Zˈ (&gt;1) crystal structures are relatively uncommon and are thought to arise through competition between intermolecular interactions of similar strength. As such high Zˈ crystal structures are challenging to predict, new examples are valuable in improving understanding in the field. Herein, we report the X-ray crystal structures of a series of shikimate esters, the asymmetric units of which exhibit high Zˈ values. Of special interest is the crystal structure of methyl shikimate, the asymmetric unit of which comprises 12 independent molecules; Zˈ = 12. This uncommonly large Zˈ value arises through a combination of factors, including the intrinsic homochirality of the molecule, the conformational inflexibility of the cyclohexene ring, the presence of multiple hydrogen bonding motifs, and both the cis- and trans-conformers of the ester moiety. Comparison of the X-ray crystal structures of shikimic acid, methyl shikimate, ethyl shikimate, and iso-propyl shikimate suggests that instances of high Zˈ in this series correlate with specific hydrogen bonding motifs influenced by the steric bulk of the ester. The results of this study provide important insights into factors that influence the formation of organic crystal structures where the value of Zˈ is greater than 1

High-Throughput Encapsulated Nanodroplet Screening for Accelerated Co-Crystal Discovery

Co-crystals are composed of two or more chemically inequivalent molecular species, excluding solvents, generally in a stoichiometric ratio. Co-crystals are particularly important in pharmaceutical development, where a suitable co-crystal can significantly improve the physiochemical and pharmacokinetic properties of an active pharmaceutical ingredient. However, co-crystal discovery remains both practically challenging and resource intensive, requiring the extensive searching of complex experimental space. Herein, we demonstrate a high-throughput (HTP) nanoscale co-crystallisation method for the rapid screening of large areas of co-crystallisation space with minimal sample requirements, based on Encapsulated Nanodroplet Crystallisation (ENaCt). HTP co-crystallisation screening by ENaCt allowed rapid access to all 18 possible binary co-crystal combinations of 3 small molecules and 6 co-formers (A/B), through the use of 3,456 individual experiments exploring solvent, encapsulating oil and stoichiometry, including 10 novel binary co-crystal structures elucidated by single crystal X-ray diffraction (SCXRD). Higher-order co-crystal (HOC) discovery, accessing co-crystals containing three or more molecules, is one of the most challenging co-crystal research areas, due to the highly complex experimental landscape that must be navigated. Herein, we further exemplify the power of ENaCt co-crystallisation by applicaton to HOC discovery. HTP ENaCt co-crystallisation screening of three component (A/B/C) and four component (A/B/C/D) combinations gave ready access to both ternary and quaternary HOCs, each containing three or four different molecular species respectively. In total, 13,056 individual ENaCt experiments are presented resulting in 54 co-crystal structures by SCXRD, including 17 novel binary co-crystals, 8 novel ternary co-crystals and 4 novel quaternary co-crystals. ENaCt co-crystallisation is thus demonstrated to be a highly impactful and efficient tool in the search for small molecule co-crystals, through the employment of parallelised HTP nanoscale experimental workflows

Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

Citation: Garcia, B. L., Zhi, H., Wager, B., Hook, M., & Skare, J. T. (2016). Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex. Plos Pathogens, 12(1), 28. doi:10.1371/journal.ppat.1005404Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems

Alkali Metal Complexes of Phosphine-Borane-Substituted Benzyl Ligands and Their Application in the Synthesis of B-H\ub7\ub7\ub7Sn Stabilized Dialkylstannylenes

\ua9 2024 The Authors. Published by American Chemical Society.The benzyl-substituted phosphine-boranes PhCH2P(BH3)R2 [R = iPr (1H), Ph (2H), Cy (3H)] are accessible through either the reaction between R2PCl and PhCH2MgBr, followed by treatment with BH3\ub7SMe2 or the reaction between R2P(BH)3Li and PhCH2Br. Treatment of 1H, 2H, or 3H with nBuLi, PhCH2Na, or PhCH2K gave the corresponding alkali metal complexes [{iPr2P(BH3)CHPh}Li(THF)]2 (1Li), [{Ph2P(BH3)CHPh}Li(OEt2)2] (2Li), [{Cy2P(BH3)CHPh}Li(TMEDA)] (3Li), [iPr2P(BH3)CHPh]Na (1Na), [{Ph2P(BH3)CHPh}Na(THF)2]2 (2Na), [Cy2P(BH3)CHPh]Na(THF)0.5 (3Na), [{iPr2P(BH3)CHPh}K]∞ (1K), [{Ph2P(BH3)CHPh}K(THF)]∞ (2K), and [{Cy2P(BH3)CHPh}K.0.5PhMe]∞ (3K). X-ray crystallography revealed that, while 2Li and 3Li crystallize as monomers, 1Li and 2Na crystallize as borane-bridged dimers. The potassium complexes 1K, 2K, and 3K all crystallize with polymeric structures, in which the monomer units are linked to each other through a range of both bridging BH3 groups and multihapto interactions between the potassium cations and the aromatic rings. The reactions between two equivalents of either 1Li or 3Li and Cp2Sn gave the corresponding dialkylstannylenes [{R2P(BH3)CHPh}2Sn] [R = iPr (1Sn), Cy (3Sn)]. These compounds were isolated as mixtures of the rac and meso diastereomers. X-ray crystallography reveals that rac-1Sn and rac-3Sn crystallize as discrete monomers each exhibiting two agostic-type B-H\ub7\ub7\ub7Sn contacts

RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord

ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned \u3e50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG’s). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNFα-induced protein 2 (TNFAIP2) as a major network “hub” gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF’s involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNFα signaling may be helpful in ALS patients

TNFR1 inhibition with a nanobody protects against EAE development in mice

TNF has as detrimental role in multiple sclerosis (MS), however, anti-TNF medication is not working. Selective TNF/TNFR1 inhibition whilst sparing TNFR2 signaling reduces the pro-inflammatory effects of TNF but preserves the important neuroprotective signals via TNFR2. We previously reported the generation of a Nanobody-based selective inhibitor of human TNFR1, TROS that will be tested in experimental autoimmune encephalomyelitis (EAE). We specifically antagonized TNF/TNFR1 signaling using TROS in a murine model of MS, namely MOG(35-55)-induced EAE. Because TROS does not cross-react with mouse TNFR1, we generated mice expressing human TNFR1 in a mouse TNFR1-knockout background (hTNFR1 Tg), and we determined biodistribution of Tc-99m-TROS and effectiveness of TROS in EAE in those mice. Biodistribution analysis demonstrated that intraperitoneally injected TROS is retained more in organs of hTNFR1 Tg mice compared to wild type mice. TROS was also detected in the cerebrospinal fluid (CSF) of hTNFR1 Tg mice. Prophylactic TROS administration significantly delayed disease onset and ameliorated its symptoms. Moreover, treatment initiated early after disease onset prevented further disease development. TROS reduced spinal cord inflammation and neuroinflammation, and preserved myelin and neurons. Collectively, our data illustrate that TNFR1 is a promising therapeutic target in MS

Studies on the Epipolythiodioxopiperazine Alkaloid Verticillin D: Scaled Production, Streamlined Purification, and Absolute Configuration<strong> </strong>

Verticillins, epipolythiodioxopiperazine alkaloids that were first described over 50 years ago, have undergone extensive cytotoxic and pharmacological evaluations over the last decade. However, of the 27 verticillin analogues in the literature, the chemistry of verticillin D, which has two additional secondary hydroxy moieties, relative to verticillin A, has remained largely unexplored since its discovery in 1999. With the goal of advancing our understanding of verticillin D, there were three main objectives with this study: improving production, streamlining purification, and assigning absolute configuration via X-ray crystallography. To begin, the production of verticillin D was analyzed across seven fungal strains, and the top producer was further assessed under two fermentation conditions. Clonostachys rosea (strain MSX51257) biosynthesized the highest amount of verticillin D, with production peaking between 15-25 days on rice media. Interestingly, in contrast to similar studies that yield verticillin A, the biosynthesis of verticillin D was not accompanied by a suite of structurally related verticillin analogues. As such, the purification of verticillin D was more rapid and could be accomplished without the use of HPLC. These materials were used, in part, to determine the absolute configuration of verticillin D via X-ray crystallography, allowing for assignment of the asymmetric centers at both the 13 and 13′ positions as R, which has never been accomplished. This is only the third report of an X-ray structure of a verticillin analogue

BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin Α3Β1

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function