In-Plate Chemical Synthesis of Isopeptide-Linked SUMOylated Peptide Fluorescence Polarization Reagents for High-Throughput Screening of SENP Preferences.

Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83 isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1-3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide-linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel-based read-out.

Total Chemical Synthesis of a Functionalized GFP Nanobody.

Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional in vitro validation in pull-down and confocal microscopy settings.

Solid-phase synthesis and biological evaluation of piperazine-based novel bacterial topoisomerase inhibitors.

There is an emerging global need for new and more effective antibiotics against multi-resistant bacteria. This situation has led to massive industrial investigations on novel bacterial topoisomerase inhibitors (NBTIs) that target the vital bacterial enzymes DNA gyrase and topoisomerase IV. However, several of the NBTI compound classes have been associated with inhibition of the hERG potassium channel, an undesired cause of cardiac arrhythmia, which challenges medicinal chemistry efforts through lengthy synthetic routes. We herein present a solid-phase strategy that rapidly facilitates the chemical synthesis of a promising new class of NBTIs. A proof-of-concept library was synthesized with the ability to modulate both hERG affinity and antibacterial activity through scaffold substitutions.

Reactivity and Synthetic Applications of Multicomponent Petasis Reactions.

The Petasis boron-Mannich reaction, simply referred to as the Petasis reaction, is a powerful multicomponent coupling reaction of a boronic acid, an amine, and a carbonyl derivative. Highly functionalized amines with multiple stereogenic centers can be efficiently accessed via the Petasis reaction with high levels of both diastereoselectivity and enantioselectivity. By drawing attention to examples reported in the past 8 years, this Review demonstrates the breadth of the reactivity and synthetic applications of Petasis reactions in several frontiers: the expansion of the substrate scope in the classic three-component process; nonclassic Petasis reactions with additional components; Petasis-type reactions with noncanonical substrates, mechanism, and products; new asymmetric versions assisted by chiral catalysts; combinations with a secondary or tertiary transformation in a cascade- or sequence-specific manner to access structurally complex, natural-product-like heterocycles; and the synthesis of polyhydroxy alkaloids and biologically interesting molecules.

Disulphide-mediated site-directed modification of proteins.

Methods for chemical modification of native proteins in a controlled fashion are in high demand. Here, a novel protocol that exploits bifunctional reagents for transient targeting of solvent exposed disulphides to direct the introduction of a single exogenous reactive thiol handle at a lysine side chain has been developed. The protocol has successfully been applied to functionalize six different Fabs and human growth hormone.

Interaction Between the a3 Region of Factor VIII and the TIL'E' Domains of the von Willebrand Factor.

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.

Scaffold Diversity from N-Acyliminium Ions.

N-Acyliminium ions are powerful reactive species for the formation of carbon-carbon and carbon-heteroatom bonds. Strategies relying on intramolecular reactions of N-acyliminium intermediates, also referred to as N-acyliminium ion cyclization reactions, have been employed for the construction of structurally diverse scaffolds, ranging from simple bicyclic skeletons to complex polycyclic systems and natural-product-like compounds. This review aims to provide an overview of cyclization reactions of N-acyliminium ions derived from various precursors for the assembly of structurally diverse scaffolds, covering the literature over the past 12 years (from 2004 to 2015).

Peptide-Directed DNA-Templated Protein Labelling for The Assembly of a Pseudo-IgM.

The development of methods for conjugation of DNA to proteins is of high relevance for the integration of protein function and DNA structures. Here, we demonstrate that protein-binding peptides can direct a DNA-templated reaction, selectively furnishing DNA-protein conjugates with one DNA label. Quantitative conversion of oligonucleotides is achieved at low stoichiometries and the reaction can be performed in complex biological matrixes, such as cell lysates. Further, we have used a star-like pentameric DNA nanostructure to assemble five DNA-Rituximab conjugates, made by our reported method, into a pseudo-IgM antibody structure that was subsequently characterized by negative-stain transmission electron microscopy (nsTEM) analysis.

Petasis three-component reactions for the synthesis of diverse heterocyclic scaffolds.

The Petasis three-component reaction (PR) of hydroxy aldehydes, amines and boronic acids is an important multi-component reaction for the synthesis of structurally diverse scaffolds and biologically interesting small molecules. The reaction has been significantly explored in the past decade, and many new variants have emerged, such as asymmetric, traceless and four-component approaches. The excellent stereoselectivity, high yield and broad functional group tolerance altogether make this reaction ideal for fragment and compound collection synthesis, since orthogonal chemical handles can be incorporated for subsequent scaffold formation and appendage modification. Herein we present a selection of recent variations on the PR theme for the synthesis of scaffolds of relevance to medicinal chemistry.

Repurposing the anticancer drug cisplatin with the aim of developing novel Pseudomonas aeruginosa infection control agents.

Antibiotic resistance threatens effective treatment of microbial infections globally. This situation has spurred the hunt for new antimicrobial compounds in both academia and the pharmaceutical industry. Here, we report how the widely used antitumor drug cisplatin may be repurposed as an effective antimicrobial against the nosocomial pathogen Pseudomonas aeruginosa. Cisplatin was found to effectively kill strains of P. aeruginosa. In such experiments, transcriptomic profiling showed upregulation of the recA gene, which is known to be important for DNA repair, implicating that cisplatin could interfere with DNA replication in P. aeruginosa. Cisplatin treatment significantly repressed the type III secretion system (T3SS), which is important for the secretion of exotoxins. Furthermore, cisplatin was also demonstrated to eradicate in vitro biofilms and in vivo biofilms in a murine keratitis model. This showed that cisplatin could be effectively used to eradicate biofilm infections which were otherwise difficult to be treated by conventional antibiotics. Although cisplatin is highly toxic for humans upon systemic exposure, a low toxicity was demonstrated with topical treatment. This indicated that higher-than-minimal inhibitory concentration (MIC) doses of cisplatin could be topically applied to treat persistent and recalcitrant P. aeruginosa infections.

The anti-cancerous drug doxorubicin decreases the c-di-GMP content in Pseudomonas aeruginosa but promotes biofilm formation.

Current antibiotic treatments are insufficient in eradicating bacterial biofilms, which represent the primary cause of chronic bacterial infections. Thus, there is an urgent need for new strategies to eradicate biofilm infections. The second messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria. It is hypothesized that drugs lowering the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. To identify compounds capable of lowering c-di-GMP levels in Pseudomonas aeruginosa, we screened 5000 compounds for their potential c-di-GMP-lowering effect using a recently developed c-di-GMP biosensor strain. Our screen identified the anti-cancerous drug doxorubicin as a potent c-di-GMP inhibitor. In addition, the drug decreased the transcription of many biofilm-related genes. However, despite its effect on the c-di-GMP content in P. aeruginosa, doxorubicin was unable to inhibit biofilm formation or disperse established biofilms. On the contrary, the drug was found to promote P. aeruginosa biofilm formation, possibly through release of extracellular DNA from a subpopulation of killed bacteria. Our findings emphasize that lowering of the c-di-GMP content in bacteria might not be sufficient to mediate biofilm inhibition or dispersal.

A metal-catalyzed enyne-cyclization step for the synthesis of bi- and tricyclic scaffolds amenable to molecular library production.

A facile metal-catalyzed diversification step for the synthesis of novel bi- and tricyclic scaffolds from enyne substrates is reported in this study. From a single starting material, topologically diverse scaffolds for library synthesis can be generated and decorated in a few steps. The methodology was used to produce a library of 490 compounds within the European Lead Factory (ELF) Consortium.

In-Bead Screening of Hydroxamic Acids for the Identification of HDAC Inhibitors.

A one bead-one compound screening format is presented. Following solid-phase synthesis on a photolabile linker, library compounds were readily released and screened inside polymer beads. The release of screening compounds was readily controlled by varying photolysis time and light intensity. Dose-response experiments were carried out to effectively distinguish high- and low-affinity ligands. A library containing 55,800 compounds was synthesized and screened in a fluorometric assay, thereby identifying potent HDAC inhibitors with IC50 values in the nanomolar range.

Synthesis of hexahydropyrrolo[2,1-a]isoquinoline compound libraries through a Pictet-Spengler cyclization/metal-catalyzed cross coupling/amidation sequence.

Molecular libraries of natural product-like and structurally diverse compounds are attractive in early drug discovery campaigns. In here, we present synthetic methodology for library production of hexahydropyrrolo[2,1-a]isoquinoline (HPIQ) compounds. Two advanced HPIQ intermediates, both incorporating two handles for diversification, were synthesized through an oxidative cleavage/Pictet-Spengler reaction sequence in high overall yields. A subsequent metal-catalyzed cross coupling/amidation protocol was developed and its utility in library synthesis was validated by construction of a 20-membered natural product-like molecular library in good overall yields.

Synthesis of 1,4,5 trisubstituted γ-lactams via a 3-component cascade reaction.

A three component one-pot cascade reaction was developed for the synthesis of 1,4,5-trisubstituted γ-lactams. The resulting scaffold can be modified independently at three positions, two of which are conveniently accessed by changing the components of the one-pot reaction. The phases of building block generation, scaffold synthesis and subsequent appendage modification were adapted to library production, which resulted in a screening library of 500 compounds.

Triazole-containing N-acyl homoserine lactones targeting the quorum sensing system in Pseudomonas aeruginosa.

In an attempt to devise new antimicrobial treatments for biofilm infections, the bacterial cell-cell communication system termed quorum sensing has emerged as an attractive target. It has proven possible to intercept the communication system by synthetic non-native ligands and thereby lower the pathogenesis and antibiotic tolerance of a bacterial biofilm. To identify the structural elements important for antagonistic or agonistic activity against the Pseudomonas aeruginosa LasR protein, we report the synthesis and screening of new triazole-containing mimics of natural N-acyl homoserine lactones. A series of azide- and alkyne-containing homoserine lactone building blocks was used to prepare an expanded set of 123 homoserine lactone analogues through a combination of solution- and solid-phase synthesis methods. The resulting compounds were subjected to cell-based quorum sensing screening assays, thereby revealing several bioactive compounds, including 13 compounds with antagonistic activity and 9 compounds with agonistic activity.

A Four-Component Reaction for the Synthesis of Dioxadiazaborocines.

A four-component reaction for the synthesis of heterocyclic boronates is reported. Readily available hydrazides, α-hydroxy aldehydes, and two orthogonally reactive boronic acids are combined in a single step to give structurally distinct bicyclic boronates, termed dioxadiazaborocines (DODA borocines). In this remarkable process, one boronic acid reacts as a carbon nucleophile and the other as a boron electrophile to provide enantio- and diastereomerically pure heterocyclic boronates with multiple stereocenters in high yields.

Comparative systems biology analysis to study the mode of action of the isothiocyanate compound Iberin on Pseudomonas aeruginosa.

Food is now recognized as a natural resource of novel antimicrobial agents, including those that target the virulence mechanisms of bacterial pathogens. Iberin, an isothiocyanate compound from horseradish, was recently identified as a quorum-sensing inhibitor (QSI) of the bacterial pathogen Pseudomonas aeruginosa. In this study, we used a comparative systems biology approach to unravel the molecular mechanisms of the effects of iberin on QS and virulence factor expression of P. aeruginosa. Our study shows that the two systems biology methods used (i.e., RNA sequencing and proteomics) complement each other and provide a thorough overview of the impact of iberin on P. aeruginosa. RNA sequencing-based transcriptomics showed that iberin inhibits the expression of the GacA-dependent small regulatory RNAs RsmY and RsmZ; this was verified by using gfp-based transcriptional reporter fusions with the rsmY or rsmZ promoter regions. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomics showed that iberin reduces the abundance of the LadS protein, an activator of GacS. Taken together, the findings suggest that the mode of QS inhibition in iberin is through downregulation of the Gac/Rsm QS network, which in turn leads to the repression of QS-regulated virulence factors, such as pyoverdine, chitinase, and protease IV. Lastly, as expected from the observed repression of small regulatory RNA synthesis, we also show that iberin effectively reduces biofilm formation. This suggests that small regulatory RNAs might serve as potential targets in the future development of therapies against pathogens that use QS for controlling virulence factor expression and assume the biofilm mode of growth in the process of causing disease.

Solid-phase synthesis and biological evaluation of N-dipeptido L-homoserine lactones as quorum sensing activators.

Bacteria use small signaling molecules to communicate in a process termed "quorum sensing" (QS), which enables the coordination of survival strategies, such as production of virulence factors and biofilm formation. In Gram-negative bacteria, these signaling molecules are a series of N-acylated L-homoserine lactones. With the goal of identifying non-native compounds capable of modulating bacterial QS, a virtual library of N-dipeptido L-homoserine lactones was screened in silico with two different crystal structures of LasR. The 30 most promising hits were synthesized on HMBA-functionalized PEGA resin and released through an efficient acid-mediated cyclative release mechanism. Subsequent screening for modulation of QS in Pseudomonas aeruginosa and E. coli identified six moderately strong activators. A follow-up library designed from the preliminary derived structure-activity relationships was synthesized and evaluated for their ability to activate the QS system in this bacterium. This resulted in the identification of another six QS activators (two with low micromolar activity) thus illuminating structural features required for QS modulation.

Synthesis of heterocycles through transition-metal-catalyzed isomerization reactions.

Metal-catalyzed isomerization of N- and O-allylic systems is emerging as an effective method to form synthetically useful iminium and oxocarbenium intermediates. In the presence of tethered nucleophiles, several recent examples illuminate this approach as a powerful strategy for the synthesis of structurally complex and diverse heterocycles. In this Concept article, we attempt to cover this area of research through a selection of recent versatile examples.