Structure and mechanism of the ER-based glucosyltransferase ALG6.

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.

Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase.

C-linked glycosylation is essential for the trafficking, folding and function of secretory and transmembrane proteins involved in cellular communication processes. The tryptophan C-mannosyltransferase (CMT) enzymes that install the modification attach a mannose to the first tryptophan of WxxW/C sequons in nascent polypeptide chains by an unknown mechanism. Here, we report cryogenic-electron microscopy structures of Caenorhabditis elegans CMT in four key states: apo, acceptor peptide-bound, donor-substrate analog-bound and as a trapped ternary complex with both peptide and a donor-substrate mimic bound. The structures indicate how the C-mannosylation sequon is recognized by this CMT and its paralogs, and how sequon binding triggers conformational activation of the donor substrate: a process relevant to all glycosyltransferase C superfamily enzymes. Our structural data further indicate that the CMTs adopt an unprecedented electrophilic aromatic substitution mechanism to enable the C-glycosylation of proteins. These results afford opportunities for understanding human disease and therapeutic targeting of specific CMT paralogs.

Rotavirus Spike Protein VP4 Mediates Viroplasm Assembly by Association to Actin Filaments.

Rotavirus (RV) viroplasms are cytosolic inclusions where both virus genome replication and primary steps of virus progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, that contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate that rRV/VP4-BAP infection, as opposed to rRV/wt infection, did not lead to a reorganized actin cytoskeleton as viroplasms formed were insensitive to drugs that depolymerize actin and inhibit myosin. Moreover, wild-type (wt) VP4, but not VP4-BAP, appeared to associate with actin filaments. Similarly, VP4 in coexpression with NSP5 and NSP2 induced a significant increase in the number of viroplasm-like structures. Interestingly, a small peptide mimicking loop K145-G150 rescued the phenotype of rRV/VP4-BAP by increasing its ability to form viroplasms and hence improve virus progeny formation. Collectively, these results provide a direct link between VP4 and the actin cytoskeleton to catalyze viroplasm assembly. IMPORTANCE The spike protein VP4 participates in diverse steps of the rotavirus (RV) life cycle, including virus-cell attachment, internalization, modulation of endocytosis, virion morphogenesis, and virus egress. Using reverse genetics, we constructed for the first time a recombinant RV, rRV/VP4-BAP, harboring a heterologous peptide in the lectin domain (loop K145-G150) of VP4. The rRV/VP4-BAP was replication competent but with reduced fitness due to a defect in the ability to reorganize the actin cytoskeleton, which affected the efficiency of viroplasm assembly. This defect was rescued by adding a permeable small-peptide mimicking the wild-type VP4 loop K145-G150. In addition to revealing a new role of VP4, our findings suggest that rRV harboring an engineered VP4 could be used as a new dual vaccination platform providing immunity against RV and additional heterologous antigens.

Molecular Framework Analysis of the Generated Database GDB-13s.

The generated databases (GDBs) list billions of possible molecules from systematic enumeration following simple rules of chemical stability and synthetic feasibility. To assess the originality of GDB molecules, we compared their Bemis and Murcko molecular frameworks (MFs) with those in public databases. MFs result from molecules by converting all atoms to carbons, all bonds to single bonds, and removing terminal atoms iteratively until none remain. We compared GDB-13s (99,394,177 molecules up to 13 atoms containing simplified functional groups, 22,130 MFs) with ZINC (885,905,524 screening compounds, 1,016,597 MFs), PubChem50 (100,852,694 molecules up to 50 atoms, 1,530,189 MFs), and COCONUT (401,624 natural products, 42,734 MFs). While MFs in public databases mostly contained linker bonds and six-membered rings, GDB-13s MFs had diverse ring sizes and ring systems without linker bonds. Most GDB-13s MFs were exclusive to this database, and many were relatively simple, representing attractive targets for synthetic chemistry aiming at innovative molecules.

Expanding Bioactive Fragment Space with the Generated Database GDB-13s.

Identifying innovative fragments for drug design can help medicinal chemistry address new targets and overcome the limitations of the classical molecular series. By deconstructing molecules into ring fragments (RFs, consisting of ring atoms plus ring-adjacent atoms) and acyclic fragments (AFs, consisting of only acyclic atoms), we find that public databases of molecules (i.e., ZINC and PubChem) and natural products (i.e., COCONUT) contain mostly RFs and AFs of up to 13 atoms. We also find that many RFs and AFs are enriched in bioactive vs inactive compounds from ChEMBL. We then analyze the generated database GDB-13s, which enumerates 99 million possible molecules of up to 13 atoms, for RFs and AFs resembling ChEMBL bioactive RFs and AFs. This analysis reveals a large number of novel RFs and AFs that are structurally simple, have favorable synthetic accessibility scores, and represent opportunities for synthetic chemistry to contribute to drug innovation in the context of fragment-based drug discovery.

Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms.

Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.

Medium-Chain Lipid Conjugation Facilitates Cell-Permeability and Bioactivity.

The majority of bioactive molecules act on membrane proteins or intracellular targets and therefore needs to partition into or cross biological membranes. Natural products often exhibit lipid modifications to facilitate critical molecule-membrane interactions, and in many cases their bioactivity is markedly reduced upon removal of a lipid group. However, despite its importance in nature, lipid-conjugation of small molecules is not commonly used in chemical biology and medicinal chemistry, and the effect of such conjugation has not been systematically studied. To understand the composition of lipids found in natural products, we carried out a chemoinformatic characterization of the "natural product lipidome". According to this analysis, lipidated natural products predominantly contain saturated medium-chain lipids (MCLs), which are significantly shorter than the long-chain lipids (LCLs) found in membranes and lipidated proteins. To study the usefulness of such modifications in probe design, we systematically explored the effect of lipid conjugation on five different small molecule chemotypes and find that permeability, cellular retention, subcellular localization, and bioactivity can be significantly modulated depending on the type of lipid tail used. We demonstrate that MCL conjugation can render molecules cell-permeable and modulate their bioactivity. With all explored chemotypes, MCL-conjugates consistently exhibited superior uptake or bioactivity compared to LCL-conjugates and either comparable or superior uptake or bioactivity to short-chain lipid (SCL)-conjugates. Together, our findings suggest that conjugation of small molecules with MCLs could be a powerful strategy for the design of probes and drugs.

Molecular Similarity for Drug Discovery, Target Prediction and Chemical Space Visualization.

Similar drug molecules often have similar properties and activities. Therefore, quantifying molecular similarity is central to drug discovery and optimization. Here I review computational methods using molecular similarity measures developed in my group within the interdisciplinary network NCCR TransCure investigating the physiology, structural biology and pharmacology of ion channels and membrane transporters. We designed a 3D molecular shape and pharmacophore comparison algorithm to optimize weak and unselective inhibitors by scaffold hopping and discovered potent and selective inhibitors of the ion channels TRPV6 and TRPM4, of endocannabinoid membrane transport, and of the divalent metal transporters DMT1 and ZIP8. We predicted off-target effects by combining molecular similarity searches from different molecular fingerprints against target annotated compounds from the ChEMBL database. Finally, we created interactive chemical space maps reflecting molecular similarities to facilitate the selection of screening compounds and the analysis of screening results. These different tools are available online at https://gdb.unibe.ch/tools/.

Automatic Extraction of Reaction Templates for Synthesis Prediction.

Several tools for the computational planning of synthetic routes have been developed over the last 60 years. Traditionally these have been built on manually or automatically extracted reaction rules or templates obtained from a deep knowledge of organic chemistry in the case of the former, and reaction databases for the latter. Herein we give an introductory overview to the process of automatically extracting reaction templates, starting from methods for reaction centre identification, through to their use in computer aided synthesis planning and the de novo design of compounds.

Vitaport: A Learning and Artistic Path for Public Outreach on Transporter Biology, Health and Drug Discovery.

In occasion of its conclusion, the National Center of Competence in Research (NCCR) TransCure launched a temporary learning and artistic path in the city of Bern named 'Vitaport - Was unser Körper transportiert'. The path explained how nutrients are transported through our body and how molecules find their way to the right organ to achieve their effect there. NCCR TransCure researchers, together with students of the Bern School of Design, developed ceramic objects, texts and information graphics that took the public on a multidisciplinary journey of discovery through the human body. In this article, we report about aims, development, challenges and outcome of this ambitious science outreach project in which we could experience a rewarding and successful collaboration between scientists and artists.

Physiological and Molecular Function of the Sodium/Hydrogen Exchanger NHA2 (SLC9B2).

NHA2, also known as SLC9B2, is an orphan intracellular Na+/H+ exchanger (NHE) that has been associated with arterial hypertension and diabetes mellitus in humans. The objective of this NCCR TransCure project was to define the physiological and molecular function of NHA2, to develop a high resolution kinetic transport assay for NHA2 and to identify specific and potent compounds targeting NHA2. In this review, we summarize the results of this highly interdisciplinary and interfaculty effort, led by the groups of Proffs. Jean-Louis Reymond, Christoph von Ballmoos and Daniel Fuster.

Structure and mechanism of an active lipid-linked oligosaccharide flippase.

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

Antimicrobial Peptide Dendrimers and Quorum-Sensing Inhibitors in Formulating Next-Generation Anti-Infection Cell Therapy Dressings for Burns.

Multidrug resistance infections are the main cause of failure in the pro-regenerative cell-mediated therapy of burn wounds. The collagen-based matrices for delivery of cells could be potential substrates to support bacterial growth and subsequent lysis of the collagen leading to a cell therapy loss. In this article, we report the development of a new generation of cell therapy formulations with the capacity to resist infections through the bactericidal effect of antimicrobial peptide dendrimers and the anti-virulence effect of anti-quorum sensing MvfR (PqsR) system compounds, which are incorporated into their formulation. Anti-quorum sensing compounds limit the pathogenicity and antibiotic tolerance of pathogenic bacteria involved in the burn wound infections, by inhibiting their virulence pathways. For the first time, we report a biological cell therapy dressing incorporating live progenitor cells, antimicrobial peptide dendrimers, and anti-MvfR compounds, which exhibit bactericidal and anti-virulence properties without compromising the viability of the progenitor cells.

Peptide Dendrimers: From Enzyme Models to Antimicrobials and Transfection Reagents.

Aiming at studying cooperativity effects between amino acids in easily accessible protein models, we have explored the chemistry of peptide dendrimers, which we obtain as pure products by solid-phase peptide synthesis using a branching diamino acid such as lysine at every second or third position in a peptide sequence, followed by reverse-phase HPLC purification. This article reviews discoveries driven by combinatorial library synthesis and screening, including enantioselective esterase and aldolase enzyme models, cobalamin binding and peroxidase dendrimers, glycopeptide dendrimer biofilm inhibitors and their X-ray crystal structures as complexes with lectins, antimicrobial peptide dendrimers active against multidrug resistant Gram-negative bacteria, and transfection reagents for siRNA and CRISPR-Cas9 plasmid DNA. Latest developments include cheminformatics and artificial intelligence for exploring the peptide chemical space, and the principle of stereorandomization to understand the role of peptide chirality in activity.

A Potent and Selective Janus Kinase Inhibitor with a Chiral 3D-Shaped Triquinazine Ring System from Chemical Space.

The generated databases (GDBs) enumerate billions of possible molecules following simple rules of chemical stability and synthetic feasibility. Exploring the GDBs shows that many chiral, 3D-shaped ring systems, often containing quaternary centers, have never been exploited for drug design. Shown herein is that such ring systems can be useful for medicinal chemistry by using the example of the enantioselective synthesis of triquinazine, a novel chiral piperazine analogue derived from angular triquinane. It is used to design a nanomolar and selective inhibitor of Janus Kinase 1 and is related to the marketed drug Tofacitinib, which is useful for treating autoimmune diseases.

An Immunomodulatory Peptide Dendrimer Inspired from Glatiramer Acetate.

Glatiramer acetate (GA) is a random polypeptide drug used to treat multiple sclerosis (MS), a chronic autoimmune disease. With the aim of identifying a precisely defined alternative to GA, we synthesized a library of peptide dendrimers with an amino acid composition similar to GA. We then challenged the dendrimers to trigger the release of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) from human monocytes, which is one of the effects of GA on immune cells. Several of the largest dendrimers tested were as active as GA. Detailed profiling of the best hit showed that this dendrimer induces the differentiation of monocytes towards an M2 (anti-inflammatory) state as GA does, however with a distinct immune marker profile. Our peptide dendrimer might serve as starting point to develop a well-defined immunomodulatory analog of GA.

Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa.

Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

Fluorescent Peptide Dendrimers for siRNA Transfection: Tracking pH Responsive Aggregation, siRNA Binding, and Cell Penetration.

Transfecting nucleic acids into various cells is a key procedure in biological research also envisioned for therapeutic applications. In our effort to obtain simple reagents that would be readily accessible from commercial building blocks, we recently reported peptide dendrimers as single component siRNA transfection reagents accessible in pure form by solid-phase peptide synthesis. Here, we extend our studies of these dendrimers by identifying analogs bearing a coumarin or BODIPY fluorescent label in their core and displaying comparable siRNA transfection efficiencies, pH dependent aggregation, siRNA binding, and secondary structures. Fluorescence resonance energy transfer (FRET) studies show that the dendrimers are tightly associated with siRNA within the formed nanoparticles at pH 7.4 but are released into solution at pH 5.0 and can participate in endosome escape by destabilizing the membrane at this pH value. Colocalization studies furthermore suggest that peptide dendrimers and siRNA remain tightly associated throughout the transfection process.

Populating Chemical Space with Peptides Using a Genetic Algorithm.

In drug discovery, one uses chemical space as a concept to organize molecules according to their structures and properties. One often would like to generate new possible molecules at a specific location in the chemical space marked by a molecule of interest. Herein, we report the peptide design genetic algorithm (PDGA, code available at https://github.com/reymond-group/PeptideDesignGA ), a computational tool capable of producing peptide sequences of various topologies (linear, cyclic/polycyclic, or dendritic) in proximity of any molecule of interest in a chemical space defined by macromolecule extended atom-pair fingerprint (MXFP), an atom-pair fingerprint describing molecular shape and pharmacophores. We show that the PDGA generates high-similarity analogues of bioactive peptides with diverse peptide chain topologies and of nonpeptide target molecules. We illustrate the chemical space accessible by the PDGA with an interactive 3D map of the MXFP property space available at http://faerun.gdb.tools/ . The PDGA should be generally useful to generate peptides at any location in the chemical space.

Synergistic Effect of Propidium Iodide and Small Molecule Antibiotics with the Antimicrobial Peptide Dendrimer G3KL against Gram-Negative Bacteria.

There is an urgent need to develop new antibiotics against multidrug-resistant bacteria. Many antimicrobial peptides (AMPs) are active against such bacteria and often act by destabilizing membranes, a mechanism that can also be used to permeabilize bacteria to other antibiotics, resulting in synergistic effects. We recently showed that G3KL, an AMP with a multibranched dendritic topology of the peptide chain, permeabilizes the inner and outer membranes of Gram-negative bacteria including multidrug-resistant strains, leading to efficient bacterial killing. Here, we show that permeabilization of the outer and inner membranes of Pseudomonas aeruginosa by G3KL, initially detected using the DNA-binding fluorogenic dye propidium iodide (PI), also leads to a synergistic effect between G3KL and PI in this bacterium. We also identify a synergistic effect between G3KL and six different antibiotics against the Gram-negative Klebsiella pneumoniae, against which G3KL is inactive.