Exploring a De Novo Route to Bradyrhizose: Synthesis and Isomeric Equilibrium of Bradyrhizose Diastereomers≠.

A de novo asymmetric strategy for the synthesis of d-bradyrhizose diastereomers from an achiral ketoenolester precursor is described. Key transformations used in the stereodivergent approach include two Noyori asymmetric reductions, an Achmatowicz rearrangement, diastereoselective alkene oxidations, and the first example of a palladium(0)-catalyzed glycosylation of a vinylogous pyranone. The isomeric composition of the bicyclic reducing sugars obtained was analyzed and their behaviour was compared to the natural product, revealing key stereocentres that impact the overall distribution.

The RNA binding proteins ZFP36L1 and ZFP36L2 are dysregulated in airway epithelium in human and a murine model of asthma.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma. Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma. Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2. Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored. METHODS: Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation-one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery. RESULTS: Primitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation. CONCLUSIONS: Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract.

De novo asymmetric Achmatowicz approach to oligosaccharide natural products.

The development and application of the asymmetric synthesis of oligosaccharides from achiral starting materials is reviewed. This de novo asymmetric approach centers around the use of asymmetric catalysis for the synthesis of optically pure furan alcohols in conjunction with Achmatowicz oxidative rearrangement for the synthesis of various pyranones. In addition, the use of a diastereoselective palladium-catalyzed glycosylation and subsequent diastereoselective post-glycosylation transformation was used for the synthesis of oligosaccharides. The application of this approach to oligosaccharide synthesis is discussed.

Synthetic Efforts and Ultimate Limitation to an Asymmetric Achmatowicz Approach Toward EBC-23.

An effort toward the total synthesis of the polyketide natural product EBC-23 is reported. The asymmetric approach is convergent and uses a late-stage Claisen-like enolate/acid chloride coupling to establish a key 1,3-diketone intermediate. The 1,3-diketone target is an oxidized form of the hydrated natural product, which fails to spiroketalize. The convergent asymmetric synthesis uses an asymmetric Noyori transfer hydrogenation of a ß-furyl ketoester to enantioselectively form a chiral furyl alcohol. An Achmatowicz/Jones/Luche three-step reaction sequence was used to stereoselectively convert the furyl alcohol into the 5-hydroxy-pyran-2-one. The absolute stereochemistry of the 1,3-polyol fragment was established by a Leighton allylation. A subsequent Grubbs cross-metathesis, and Evans acetalation were used to install the 1,3-syn-diol stereochemistry.

Stereoselective Synthesis of ß-Glycinamide Ribonucleotide.

A diastereoselective synthesis of the ß-anomer of glycinamide ribonucleotide (ß-GAR) has been developed. The synthesis was accomplished in nine steps from D-ribose and occurred in 5% overall yield. The route provided material on the multi-milligram scale. The synthetic ß-GAR formed was remarkably resistant to anomerization both in solution and as a solid.

Functional Characterization of Structural Genomics Proteins in the Crotonase Superfamily.

Members of the Crotonase superfamily, a mechanistically diverse family of proteins that share a conserved quaternary structure, can often catalyze more than one reaction. However, the spectrum of activity for its members has not been well studied. We report on measured crotonase and hydrolase activity for eight structural genomics (SG) proteins from the Crotonase superfamily plus two previously characterized proteins, intended as controls: human enoyl CoA hydratase (ECH) and Anabaena ß-diketone hydrolase. Like most of the 15,000+ SG protein structures deposited in the Protein Data Bank (PDB), the eight SG proteins are of unknown or uncertain biochemical function. The functional characterization of the eight SG proteins is guided by the Structurally Aligned Local Sites of Activity (SALSA), a local-structure-based computational approach to functional annotation. For human ECH, the turnover number for hydrolase activity is threefold higher than that for ECH activity, although the catalytic efficiency is 160-fold higher for ECH. Three SG proteins originally annotated as ECHs were predicted by SALSA to be hydrolases and are observed to have higher catalytic efficiencies for hydrolase activity than for ECH activity, on par with the previously characterized hydrolase. Among the five SG proteins predicted by SALSA to be ECHs, all but one also show some hydrolase activity; all five exhibit lower ECH activity than the human ECH with respect to the crotonyl-CoA substrate. Here, we show examples demonstrating that SALSA can correct functional misannotations even within enzyme families that display promiscuous activity.

Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca2+ -ATPase.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

Identifying requirements for RSK2 specific inhibitors.

Identifying isoform-specific inhibitors for closely related kinase family members remains a substantial challenge. The necessity for achieving this specificity is exemplified by the RSK family, downstream effectors of ERK1/2, which have divergent physiological effects. The natural product, SL0101, a flavonoid glycoside, binds specifically to RSK1/2 through a binding pocket generated by an extensive conformational rearrangement within the RSK N-terminal kinase domain (NTKD). In modelling experiments a single amino acid that is divergent in RSK3/4 most likely prevents the required conformational rearrangement necessary for SL0101 binding. Kinetic analysis of RSK2 association with SL0101 and its derivatives identified that regions outside of the NTKD contribute to stable inhibitor binding. An analogue with an n-propyl-carbamate at the 4" position on the rhamnose moiety was identified that forms a highly stable inhibitor complex with RSK2 but not with RSK1. These results identify a SL0101 modification that will aid the identification of RSK2 specific inhibitors.

Structure-activity relationship of avocadyne.

Avocado consumption is associated with numerous health benefits. Avocadyne is a terminally unsaturated, 17-carbon long acetogenin found almost exclusively in avocados with noted anti-leukemia and anti-viral properties. In this study, specific structural features such as the terminal triple bond, odd number of carbons, and stereochemistry are shown to be critical to its ability to suppress mitochondrial fatty acid oxidation and impart selective activity in vitro and in vivo. Together, this is the first study to conduct a structure-activity analysis on avocadyne and outline the chemical moieties critical to fatty acid oxidation suppression.

Total and Formal Syntheses of Fostriecin.

Two formal syntheses and one total synthesis of fostriecin (1) have been achieved, as well as, the synthesis of its related congener dihydro-dephospho-fostriecin. All the routes use the Sharpless dihydroxylation to set the absolute stereochemistry at C-8/9 positions and a Leighton allylation to set the C-5 position of the natural product. In the formal syntheses a Noyori transfer hydrogenation of an ynone was used to set the C-11 position while the total synthesis employed a combination of asymmetric dihydroxylation and Pd-π-allyl reduction to set the C-11 position. Finally in the total synthesis, a trans-hydroboration of the C-12/13 alkyne was used in combination with a Suzuki cross coupling to establish the Z,Z,E-triene of fostriecin (1).

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Synthesis and biological study of the phomopsolide and phomopsolidone natural products.

The complete history of the syntheses and biological activities of the phomopsolide and phomopsolidone classes of natural products is reviewed. These efforts include the successful synthesis of four of the five phomopsolide natural products, two of the four phomopsolidone natural products and two analogues of phomopsolide E, including the 7-oxa and 7-aza analogues. In addition, the utility of these synthetic efforts to enable the initial structure activity relationship studies for these classes of natural products is also covered.

Differential occupational risks to healthcare workers from SARS-CoV-2 observed during a prospective observational study.

We conducted voluntary Covid-19 testing programmes for symptomatic and asymptomatic staff at a UK teaching hospital using naso-/oro-pharyngeal PCR testing and immunoassays for IgG antibodies. 1128/10,034 (11.2%) staff had evidence of Covid-19 at some time. Using questionnaire data provided on potential risk-factors, staff with a confirmed household contact were at greatest risk (adjusted odds ratio [aOR] 4.82 [95%CI 3.45-6.72]). Higher rates of Covid-19 were seen in staff working in Covid-19-facing areas (22.6% vs. 8.6% elsewhere) (aOR 2.47 [1.99-3.08]). Controlling for Covid-19-facing status, risks were heterogenous across the hospital, with higher rates in acute medicine (1.52 [1.07-2.16]) and sporadic outbreaks in areas with few or no Covid-19 patients. Covid-19 intensive care unit staff were relatively protected (0.44 [0.28-0.69]), likely by a bundle of PPE-related measures. Positive results were more likely in Black (1.66 [1.25-2.21]) and Asian (1.51 [1.28-1.77]) staff, independent of role or working location, and in porters and cleaners (2.06 [1.34-3.15]).

Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells.

BACKGROUND: Cardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes. METHODS: We treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration. RESULTS: Digitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling. CONCLUSIONS: Our study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity. SIGNIFICANCE: Discovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.

RSK2 Maintains Adult Estrogen Homeostasis by Inhibiting ERK1/2-Mediated Degradation of Estrogen Receptor Alpha.

In response to estrogens, estrogen receptor alpha (ERα), a critical regulator of homeostasis, is degraded through the 26S proteasome. However, despite the continued presence of estrogen before menopause, ERα protein levels are maintained. We discovered that ERK1/2-RSK2 activity oscillates during the estrous cycle. In response to high estrogen levels, ERK1/2 is activated and phosphorylates ERα to drive ERα degradation and estrogen-responsive gene expression. Reduction of estrogen levels results in ERK1/2 deactivation. RSK2 maintains redox homeostasis, which prevents sustained ERK1/2 activation. In juveniles, ERK1/2-RSK2 activity is not required. Mammary gland regeneration demonstrates that ERK1/2-RSK2 regulation of ERα is intrinsic to the epithelium. Reduced RSK2 and enrichment in an estrogen-regulated gene signature occur in individuals taking oral contraceptives. RSK2 loss enhances DNA damage, which may account for the elevated breast cancer risk with the use of exogenous estrogens. These findings implicate RSK2 as a critical component for the preservation of estrogen homeostasis.

New strategies and structural considerations in development of therapeutics for carbapenem-resistant Enterobacteriaceae.

Antimicrobial resistance poses a significant threat to our ability to treat infections. Especially concerning is the emergence of carbapenem-resistant Enterobacteriaceae (CRE). In the new 2019 United States Centers for Disease Control and Prevention Antibiotic Resistance Report, CRE remain in the most urgent antimicrobial resistance threat category. There is good reason for this concerning designation. In particular, the combination of several resistance elements in CRE can make these pathogens untreatable or effectively untreatable with our current armamentarium of anti-infective agents. This article reviews recently approved agents with activity against CRE and a range of modalities in the pipeline, from early academic investigation to those in clinical trials, with a focus on structural aspects of new antibiotics. Another article in this series addresses the need to incentive pharmaceutical companies to invest in CRE antimicrobial development and to encourage hospitals to make these agents available in their formularies. This article will also consider the need for change in requirements for antimicrobial susceptibility testing implementation in clinical laboratories to address practical roadblocks that impede our efforts to provide even existing CRE antibiotics to our patients.

Regioselective Synthesis of a C-4'' Carbamate,C-6'' n-Pr Substituted Cyclitol Analogue of SL0101.

An asymmetric synthesis of two analogues of SL0101 (1) has been achieved. The effort is aimed at the discovery of inhibitors of the p90 ribosomal S6 kinase (RSK) with improved bioavailability. The route relies upon the use of the Taylor catalyst to regioselectively install C-3″ acetyl or carbamate functionality. This study led to the identification of a third-generation analogue of SL0101 with a C-4″ n-Pr-carbamate and a C-3″ acetate with improved RSK inhibitory activity.

The affinity of RSK for cylitol analogues of SL0101 is critically dependent on the B-ring C-4'-hydroxy.

Five cyclitol analogues of SL0101 with variable substitution at the C-4' position (i.e., OH, Cl, F, H, OMe) were synthesized. The series of analogues were evaluated for their ability to inhibit p90 ribosomal S6 kinase (RSK) activity. The study demonstrated the importance of the B-ring C-4' hydroxy group for RSK1/2 inhibition.