Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide's affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.

Forced convection of non-darcy flow of ethylene glycol conveying copper(II) oxide and titanium dioxide nanoparticles subject to lorentz force on wedges: Non-newtonian casson model.

The topic of two-dimensional steady laminar MHD boundary layer flow across a wedge with non-Newtonian hybrid nanoliquid (CuO-TiO2/C2H6O2) with viscous dissipation and radiation is taken into consideration. The controlling partial differential equations have been converted to non-linear higher-order ordinary differential equations using the appropriate similarity transformations. It is demonstrated that a number of thermo-physical characteristics govern the transmuted model. The issue is then mathematically resolved. When the method's accuracy is compared to results that have already been published, an excellent agreement is found. While the thermal distribution increases with an increase in Eckert number, radiation and porosity parameters, the velocity distribution decreases as porosity increases.

Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair.

Developing an effective binder for a specific ubiquitin (Ub) chain is a promising approach for modulating various biological processes with potential applications in drug discovery. Here, we combine the Random Non-standard Peptides Integrated Discovery (RaPID) method and chemical protein synthesis to screen an extended library of macrocyclic peptides against synthetic Lys63-linked Di-Ub to discover a specific binder for this Ub chain. Furthermore, next-generation binders are generated by chemical modifications. We show that our potent cyclic peptide is cell-permeable, and inhibits DNA damage repair, leading to apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide supports our findings. Collectively, we establish a powerful strategy for selective inhibition of protein-protein interactions associated with Lys63-linked Di-Ub using cyclic peptides. This study offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling.

Gold(I)-Mediated Rapid Cyclization of Propargylated Peptides via Imine Formation.

In fundamental research and drug discovery, there is still a need for effective and straightforward chemical approaches for generating cyclic peptides. The divergent synthesis of cyclic peptides remains a challenge, in particular when cyclization is carried out in the presence of unprotected side chains and a nonpeptidic component within the cycle is needed. Herein, we describe a novel and efficient strategy based on Au(I)-mediated cyclization of unprotected peptides through rapid (30-60 min) amine addition on a propargyl group to generate an imine linkage. Mechanistic insights reveal that the reaction proceeds via regioselective Markovnikov's addition of the amine on the Au(I)-activated propargyl. This strategy was successfully applied to prepare efficiently (56-94%) over 35 diverse cyclic peptides having different sequences and lengths. We have also achieved stereoselective reduction of cyclic imines employing chiral ligands. The practicality of our method was extended for the synthesis of cyclic peptides that bind Lys48-linked di-ubiquitin chains with high affinity, leading to apoptosis of cancer cells.

Highly Efficient Cyclization Approach of Propargylated Peptides via Gold(I)-Mediated Sequential C-N, C-O, and C-C Bond Formation.

A rapid and efficient cyclization of unprotected N-propargylated peptides using the Au(I) organometallic complex is reported. The method relies on the activation of the propargyl functionality using gold(I) to produce a new linkage with the N-terminus amine at the cyclization site. The presented method features a fast reaction rate (within 20 min), mild conditions, chemoselectivity, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains.

Correction: In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides.

[This corrects the article DOI: 10.1039/D0CB00179A.].

In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides.

Cancer and other disease states can change the landscape of proteins post-translationally tagged with ubiquitin (Ub) chains. Molecules capable of modulating Ub chains are potential therapeutic agents, but their discovery represents a significant challenge. Recently, it was shown that de novo cyclic peptides, selected from trillion-member random libraries, are capable of binding particular Ub chains. However, these peptides were overwhelmingly proteinogenic, so the prospect of in vivo activity was uncertain. Here, we report the discovery of small, non-proteinogenic cyclic peptides, rich in non-canonical features like N-methylation, which can tightly and specifically bind Lys48-linked Ub chains. These peptides engage three Lys48-linked Ub units simultaneously, block the action of deubiquitinases and the proteasome, induce apoptosis in vitro, and attenuate tumor growth in vivo. This highlights the potential of non-proteinogenic cyclic peptide screening to rapidly find in vivo-active leads, and the targeting of ubiquitin chains as a promising anti-cancer mechanism of action.

The Development of a Fluorescence-Based Competitive Assay Enabled the Discovery of Dimeric Cyclic Peptide Modulators of Ubiquitin Chains.

Development of modulators targeting specific interactions of ubiquitin-based conjugates with their partners is a formidable task since it requires a suitable screening assay and homogeneous ubiquitin conjugates. We developed a novel high-throughput strategy for screening ligands for Lys48-linked tetraubiquitin chain in a relatively simple, fast, and affordable manner. This approach combined with a state-of-the-art toolbox of chemical protein synthesis and a specially optimized Cys deprotection protocol enabled us to design highly potent, Lys48-linked tetraubiquitin chain selective "next generation" dimeric peptide modulators. The dimeric peptide exhibited cancer cell permeability and induced cell death with higher efficiency compared to its monocyclic analogue. These features make our dimeric peptide a promising candidate for further studies using in vivo models. Our assay can be adopted for other various ubiquitin chains in their free or anchored forms as well as conjugates for Ub-like modifiers.

Anti-inflammatory and analgesic activity of Habbe Gule Aakh, A polyherbal Unani formulation in animal models.

BACKGROUND: Habbe Gule Aakh is extensively used in Unani medicine for anti-inflammatory and analgesic activity. OBJECTIVE: To evaluate anti-inflammatory and analgesic effects of Habbe Gule Aakh on Wistar rats and Swiss mice of either sex. MATERIALS AND METHODS: The study was carried out in Wistar rats for anti-inflammatory activity while Swiss mice were used for analgesic activity. In both the tests animals were divided into five groups of six animals each which served as control, standard and test groups A, B and C. For anti-inflammatory activity, method reported by Amman was followed. For analgesic activity, Koster's protocol was adapted. RESULTS: Significant (P < 0.01) reduction in the paw volume was noted in all the test groups but less than the standard drug. Mean writhes of group B and C reduced significantly (P < 0.01) demonstrating analgesic effect. CONCLUSION: The study validated the claim of Unani medicine of use of Habbe Gule Aakh in inflammation and pain. Further, phytochemical studies are needed to know the exact mechanism of action of this formulation.

On-Demand Detachment of Succinimides on Cysteine to Facilitate (Semi)Synthesis of Challenging Proteins.

The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels-Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.

Entropy optimized MHD 3D nanomaterial of non-Newtonian fluid: A combined approach to good absorber of solar energy and intensification of heat transport.

BACKGROUND: The present work provides important insights regarding three dimensional unsteady magnetohydrodynamic flow and entropy generation of micropolar Casson Cross nanofluid subject to nonlinear thermal radiation and chemical reaction. The Buongiorno's nanofluid model featured with Brownian movement and thermophoresis is considered. Realistic aspects namely convective boundary condition, viscous dissipation and joule heating are introduced. The present problem is modeled by momentum, temperature, microrotation and nanoparticles concentration equations. METHOD: The non-dimensional highly nonlinear differential equations are solved numerically via shooting iteration technique together with 4th order Runge-Kutta integration scheme. RESULTS: The current study imparts a reasonable, pragmatic and realistic approach to a good absorber of solar energy. In addition, strong and visionary profiles of velocity, microrotation, temperature, nanoparticles concentration, entropy generation rate and Bejan number for concern nanofluids are presented. Besides, intensive physical interpretation of the involved thermophycal parameters has been well-addressed. CONCLUSIONS: The present investigation shows that strengthening of Weissenberg number uplifts the axial as well transverse fluid velocities while that of Hartmann number turns out to be a reverse trend. Furthermore, heat and mass transfer rates exhibit ascending and descending trends for intensified Brownian motion and thermophoresis respectively. Improved thermal boundary layer due to the upgrading temperature ratio parameter is another outcome of the current analysis.

Palladium-Mediated Cleavage of Proteins with Thiazolidine-Modified Backbone in Live Cells.

Chemical protein synthesis and biorthogonal modification chemistries allow production of unique proteins for a range of biological studies. Bond-forming reactions for site-selective protein labeling are commonly used in these endeavors. Selective bond-cleavage reactions, however, are much less explored and still pose a great challenge. In addition, most of studies with modified proteins prepared by either total synthesis or semisynthesis have been applied mainly for in vitro experiments with very limited extension to live cells. Reported here is an approach for studying uniquely modified proteins containing a traceless cell delivery unit and palladium-based cleavable element for chemical activation, and monitoring the effect of these proteins in live cells. This approach is demonstrated for the synthesis of a caged ubiquitin-aldehyde, which was decaged for the inhibition of deubiquitinases in live cells.

De novo macrocyclic peptides that specifically modulate Lys48-linked ubiquitin chains.

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.

Halogen Substituents in the Isoquinoline Scaffold Switches the Selectivity of Inhibition between USP2 and USP7.

Deubiquitinases are important components of the protein regulatory network and, hence, constitute a tempting drug target. We report herein structure-activity relationship studies to develop halogen-substituted isoquionoline-1,3-dione-based inhibitors of the deubiquitinase USP2. In contrast to our previous reports, the best compound discovered was found to act through a reactive oxygen species independent, uncompetitive mechanism with an IC50 of 250 nm. We show the crucial role of halogens in the common scaffold to provide potency and selectivity of our compound, where the introduction of the fluorine atom completely switches the selectivity of the inhibitor between USP2 and USP7. Our cellular studies highlight the potential applicability of the reported compound for in vivo experiments. The discovery of the isoquinoline-1,3-dione core and the knowledge obtained with regard to halogen substituents provide a platform towards understanding USP2 inhibition and the development of highly selective next-generation deubiquitinase inhibitors.

Organocatalytic decarboxylative aldol reaction of ß-ketoacids with α-ketophosphonates en route to the enantioselective synthesis of tertiary α-hydroxyphosphonates.

The first example of an asymmetric organocatalyzed decarboxylative aldol reaction of ß-ketoacids (aroylacetic acids) with α-ketophosphonates that produces a quaternary chiral centre has been developed. A quinidine based bifunctional urea derivative was identified as the preferred catalyst affording γ-aroyl tertiary α-hydroxyphosphonates in good yield and enantioselectivity. The 31P NMR spectroscopic study was performed to shed light on the reaction mechanism.

Orbital driven impurity spin effect on the magnetic order of quasi-3D cupric oxide.

Density functional calculations are performed to study the magnetic order of the severely distorted square planar cupric oxide (CuO) and local spin disorder in it in the presence of the transition metal impurities M (=Cr, Mn, Fe, Co and Ni). The distortion in the crystal structure, arisen to reduce the band energy by minimizing the covalent interaction, creates two crisscrossing zigzag spin-1/2 chains. From the spin dimer analysis we find that while the spin chain along [Formula: see text] has strong Heisenberg type antiferromagnetic coupling (J ~ 127 meV), along [Formula: see text] it exhibits weak, but robust, ferromagnetic coupling (J ~ 9 meV) mediated by reminiscent p-d covalent interactions. The impurity effect on the magnetic ordering is independent of M and purely orbital driven. If the given spin-state of M is such that the [Formula: see text] orbital is spin-polarized, then the original long-range ordering is maintained. However, if [Formula: see text] orbital is unoccupied, the absence of corresponding covalent interaction breaks the weak ferromagnetic coupling and a spin-flip takes place at the impurity site leading to breakdown of the long range magnetic ordering.

"On Water" Organocatalyzed [4 + 2] Cycloaddition of Enones and Nitro Dienes for the Enantioselective Synthesis of Densely Substituted Cyclohexanones.

An "on water" hydroquinine-based primary amine-benzoic acid organocatalyst system was found to be best suited to produce 3,4,5-trisubstituted cyclohexanones with a nitro group in the 4-position from enones and nitro dienes under ambient conditions in good yield, with good diastereoselectivity, and with excellent enantioselectivity. An appreciable rate enhancement by water was observed compared to organic solvents. Mechanistic analysis of the reaction suggests that it followed an endo [4 + 2] cycloaddition with enamine of enone as diene and nitro diene as dienophile.

Brote de Stenotrophomonas maltophilia en una UCI médica asociado a la contaminación de los grifos. Hallazgo casual de jabones contaminados con Raoultella planticola / No disponible

INTRODUCCIÓN: Stenotrophomonas maltophilia origina raramente brotes nosocomiales. MÉTODOS: Descripción de un brote y la intervención realizada. Se comparó la incidencia acumulada (IA) en el periodo epidémico (PE) y preepidémico (PPE). Se realizaron cultivos ambientales. RESULTADOS: Ocurrieron 5 casos durante el PE con una IA de 3% (IC 95% 0,4-6), 4,8 veces superior a la del PPE. S. maltophilia creció en 6 grifos y el brote se controló tras su retirada. CONCLUSIÓN: desde el punto de vista de la prevención, es importante controlar determinados reservorios para evitar brotes nosocomiales

INTRODUCTION: Stenotrophomonas maltophilia is an infrequent cause of nosocomial outbreaks. METHODS: Outbreak report and intervention study. The accumulated incidence (AI) of the epidemic period (EP) was compared with AI of the pre-epidemic period (PEP). RESULTS: During EP, five cases were identified. AI was 3% (IC 95% 0,4-6), 4,8 times higher than the AI of PEP. S. maltophilia was isolated from the tap water faucets. The outbreak was controlled after removing the faucets. CONCLUSION: It is important to control environmental reservoirs to prevent outbreaks

Contamination of liquid soap for hospital use with Raoultella planticola.

This article reports the contamination of a batch of liquid soap for hospital use with Raoultella planticola. The micro-organism was first identified as Klebsiella pneumoniae due to the inability of automated systems to characterize this species. There is a need to strengthen the inspection of cosmetic products to be used in the hospital setting. It is recommended that hospitalized patients at the highest risk of infection should use antimicrobial soaps for personal hygiene. The incidence of infections due to R. planticola is unknown as it is usually misclassified as Klebsiella spp. by automated systems.

Evaluation of in-vitro antibacterial activity and anti-inflammatory activity for different extracts of Rauvolfia tetraphylla L. root bark

To assess the in-vitro antibacterial activity and anti-inflammatory activity of orally administered different extracts (Hydro-alcoholic, methanolic, ethyl acetate and hexane) of Rauvolfia tetraphylla (R. tetraphylla) root bark in Carrageenan induced acute inflammation in rats. Methods: In-vitro antibacterial activity was evaluated for extracts against four Gram positive and four Gram negative bacteria by using cylinder plate assay. Hydro-alcoholic extract (70% v/v ethanol) at 200, 400 and 800 mg/kg doses and methanolic, ethyl acetate and hexane extracts at doses 100, 200 and 400 mg/kg were tested for anti-inflammatory activity in Carrageenan induced rat paw oedema model and paw thickness was measured every one hour up to 6 hrs. Results: All extracts of R. tetraphylla root bark showed good zone of inhibition against tested bacterial strains. In Carrageenan induced inflammation model, hydro-alcoholic and methanolic extract of R. tetraphylla root bark at three different doses produced significant (P<0.001) reduction when compared to vehicle treated control group and hexane, ethyl acetate extracts. Conclusions:In the present study extracts of R. tetraphylla root bark shows good in-vitro antibacterial activity and in-vivo anti-inflammatory activity in rats.