TurboID mapping reveals the exportome of secreted intrinsically disordered proteins in the transforming parasite Theileria annulata.

Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1-4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.

One chiral fingerprint to find them all.

Molecular fingerprints are indispensable tools in cheminformatics. However, stereochemistry is generally not considered, which is problematic for large molecules which are almost all chiral. Herein we report MAP4C, a chiral version of our previously reported fingerprint MAP4, which lists MinHashes computed from character strings containing the SMILES of all pairs of circular substructures up to a diameter of four bonds and the shortest topological distance between their central atoms. MAP4C includes the Cahn-Ingold-Prelog (CIP) annotation (R, S, r or s) whenever the chiral atom is the center of a circular substructure, a question mark for undefined stereocenters, and double bond cis-trans information if specified. MAP4C performs slightly better than the achiral MAP4, ECFP and AP fingerprints in non-stereoselective virtual screening benchmarks. Furthermore, MAP4C distinguishes between stereoisomers in chiral molecules from small molecule drugs to large natural products and peptides comprising thousands of diastereomers, with a degree of distinction smaller than between structural isomers and proportional to the number of chirality changes. Due to its excellent performance across diverse molecular classes and its ability to handle stereochemistry, MAP4C is recommended as a generally applicable chiral molecular fingerprint. SCIENTIFIC CONTRIBUTION: The ability of our chiral fingerprint MAP4C to handle stereoisomers from small molecules to large natural products and peptides is unprecedented and opens the way for cheminformatics to include stereochemistry as an important molecular parameter across all fields of molecular design.

Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Highly Potent Cationic Chitosan Derivatives Coupled to Antimicrobial Peptide Dendrimers to Combat Pseudomonas aeruginosa.

The burden of bacterial wound infections has considerably increased due to antibiotic resistance to most of the currently available antimicrobial drugs. Herein, for the first time, a chemical coupling of two cationic N-aryl (pyridyl and aminocinnamyl) chitosan derivatives to antimicrobial peptide dendrimers (AMPDs) of different generations (first, second, and third) via thioether-haloacetyl reaction is reported. The new chitosan-AMPD conjugates show high selectivity by killing Pseudomonas aeruginosa and very low toxicity toward mammalian cells, as well as extremely low hemolysis to red blood cells. Electron microscopy reveals that the new chitosan derivatives coupled to AMPD destroy both the inner and outer membranes of Gram-negative P. aeruginosa. Moreover, chitosan-AMPD conjugates show synergetic effects within extremely low concentrations. The new chitosan-AMPD conjugates can be used as potent antimicrobial therapeutic agents, to eradicate pathogens such as those present in acute and chronic infected wounds.

Antimicrobial Peptide-Peptoid Hybrids with and without Membrane Disruption.

Among synthetic analogues of antimicrobial peptides (AMPs) under investigation to address antimicrobial resistance, peptoids (N-alkylated oligoglycines) have been reported to act both by membrane disruption and on intracellular targets. Here we gradually introduced peptoid units into the membrane-disruptive undecapeptide KKLLKLLKLLL to test a possible transition toward intracellular targeting. We found that selected hybrids containing up to five peptoid units retained the parent AMP's α-helical folding, membrane disruption, and antimicrobial effects against Gram-negative bacteria including multidrug-resistant (MDR) strains of Pseudomonas aeruginosa and Klebsiella pneumoniae while showing reduced hemolysis and cell toxicities. Furthermore, some hybrids containing as few as three peptoid units as well as the full peptoid lost folding, membrane disruption, hemolysis, and cytotoxicity but displayed strong antibacterial activity under dilute medium conditions typical for proline-rich antimicrobial peptides (PrAMPs), pointing to intracellular targeting. These findings parallel previous reports that partially helical amphiphilic peptoids are privileged oligomers for antibiotic development.

Alchemical analysis of FDA approved drugs.

Chemical space maps help visualize similarities within molecular sets. However, there are many different molecular similarity measures resulting in a confusing number of possible comparisons. To overcome this limitation, we exploit the fact that tools designed for reaction informatics also work for alchemical processes that do not obey Lavoisier's principle, such as the transmutation of lead into gold. We start by using the differential reaction fingerprint (DRFP) to create tree-maps (TMAPs) representing the chemical space of pairs of drugs selected as being similar according to various molecular fingerprints. We then use the Transformer-based RXNMapper model to understand structural relationships between drugs, and its confidence score to distinguish between pairs related by chemically feasible transformations and pairs related by alchemical transmutations. This analysis reveals a diversity of structural similarity relationships that are otherwise difficult to analyze simultaneously. We exemplify this approach by visualizing FDA-approved drugs, EGFR inhibitors, and polymyxin B analogs.

Multistep retrosynthesis combining a disconnection aware triple transformer loop with a route penalty score guided tree search.

Computer-aided synthesis planning (CASP) aims to automatically learn organic reactivity from literature and perform retrosynthesis of unseen molecules. CASP systems must learn reactions sufficiently precisely to propose realistic disconnections, while avoiding overfitting to leave room for diverse options, and explore possible routes such as to allow short synthetic sequences to emerge. Herein we report an open-source CASP tool proposing original solutions to both challenges. First, we use a triple transformer loop (TTL) predicting starting materials (T1), reagents (T2), and products (T3) to explore various disconnection sites defined by combining systematic, template-based, and transformer-based tagging procedures. Second, we integrate TTL into a multistep tree search algorithm (TTLA) prioritizing sequences using a route penalty score (RPScore) considering the number of steps, their confidence score, and the simplicity of all intermediates along the route. Our approach favours short synthetic routes to commercial starting materials, as exemplified by retrosynthetic analyses of recently approved drugs.

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.

Organic Catalytic Activity as a Method for Agnostic Life Detection.

An ideal life detection instrument would have high sensitivity but be insensitive to abiotic processes and would be capable of detecting life with alternate molecular structures. In this study, we propose that catalytic activity can be the basis of a nearly ideal life detection instrument. There are several advantages to catalysis as an agnostic life detection method. Demonstrating catalysis does not necessarily require culturing/growing the alien life and in fact may persist even in dead biomass for some time, and the amplification by catalysis is large even by minute amounts of catalysts and, hence, can be readily detected against abiotic background rates. In specific, we propose a hydrolytic catalysis detection instrument that could detect activity in samples of extraterrestrial organic material from unknown life. The instrument uses chromogenic assay-based detection of various hydrolytic catalytic activities, which are matched to corresponding artificial substrates having the same, chromogenic (preferably fluorescent) upon release, group; D- and L-enantiomers of these substrates can be used to also answer the question whether unknown life is chiral. Since catalysis is a time-proportional product-concentration amplification process, hydrolytic catalytic activity can be measured on a sample of even a minute size, and with instruments based on, for example, optofluidic chip technology.

To Fold or Not to Fold: Diastereomeric Optimization of an α-Helical Antimicrobial Peptide.

Membrane disruptive α-helical antimicrobial peptides (AMPs) offer an opportunity to address multidrug resistance; however, most AMPs are toxic and unstable in serum. These limitations can be partly overcome by introducing D-residues, which often confers protease resistance and reduces toxicity without affecting antibacterial activity, presumably due to lowered α-helicity. Here, we investigated 31 diastereomers of the α-helical AMP KKLLKLLKLLL. Three diastereomers containing two, three, and four D-residues showed increased antibacterial effects, comparable hemolysis, reduced toxicity against HEK293 cells, and excellent serum stability, while another diastereomer with four D-residues additionally displayed lower hemolysis. X-ray crystallography confirmed that high or low α-helicity as measured by circular dichroism indicated α-helical or disordered structures independently of the number of chirality switched residues. In contrast to previous reports, α-helicity across diastereomers correlated with both antibacterial activity and hemolysis and revealed a complex relationship between stereochemistry, activity, and toxicity, highlighting the potential of diastereomers for property optimization.

Dipropylamine for 9-Fluorenylmethyloxycarbonyl (Fmoc) Deprotection with Reduced Aspartimide Formation in Solid-Phase Peptide Synthesis.

Herein, we report dipropylamine (DPA) as a fluorenylmethyloxycarbonyl (Fmoc) deprotection reagent to strongly reduce aspartimide formation compared to piperidine (PPR) in high-temperature (60 °C) solid-phase peptide synthesis (SPPS). In contrast to PPR, DPA is readily available, inexpensive, low toxicity, and nonstench. DPA also provides good yields in SPPS of non-aspartimide-prone peptides and peptide dendrimers.

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.

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.

Molecular basis for glycan recognition and reaction priming of eukaryotic oligosaccharyltransferase.

Oligosaccharyltransferase (OST) is the central enzyme of N-linked protein glycosylation. It catalyzes the transfer of a pre-assembled glycan, GlcNAc2Man9Glc3, from a dolichyl-pyrophosphate donor to acceptor sites in secretory proteins in the lumen of the endoplasmic reticulum. Precise recognition of the fully assembled glycan by OST is essential for the subsequent quality control steps of glycoprotein biosynthesis. However, the molecular basis of the OST-donor glycan interaction is unknown. Here we present cryo-EM structures of S. cerevisiae OST in distinct functional states. Our findings reveal that the terminal glucoses (Glc3) of a chemo-enzymatically generated donor glycan analog bind to a pocket formed by the non-catalytic subunits WBP1 and OST2. We further find that binding either donor or acceptor substrate leads to distinct primed states of OST, where subsequent binding of the other substrate triggers conformational changes required for catalysis. This alternate priming allows OST to efficiently process closely spaced N-glycosylation sites.

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening.

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action.

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.

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.

Machine Learning Guided Discovery of Non-Hemolytic Membrane Disruptive Anticancer Peptides.

Most antimicrobial peptides (AMPs) and anticancer peptides (ACPs) fold into membrane disruptive cationic amphiphilic α-helices, many of which are however also unpredictably hemolytic and toxic. Here we exploited the ability of recurrent neural networks (RNN) to distinguish active from inactive and non-hemolytic from hemolytic AMPs and ACPs to discover new non-hemolytic ACPs. Our discovery pipeline involved: 1) sequence generation using either a generative RNN or a genetic algorithm, 2) RNN classification for activity and hemolysis, 3) selection for sequence novelty, helicity and amphiphilicity, and 4) synthesis and testing. Experimental evaluation of thirty-three peptides resulted in eleven active ACPs, four of which were non-hemolytic, with properties resembling those of the natural ACP lasioglossin III. These experiments show the first example of direct machine learning guided discovery of non-hemolytic ACPs.

Reaction classification and yield prediction using the differential reaction fingerprint DRFP.

Predicting the nature and outcome of reactions using computational methods is a crucial tool to accelerate chemical research. The recent application of deep learning-based learned fingerprints to reaction classification and reaction yield prediction has shown an impressive increase in performance compared to previous methods such as DFT- and structure-based fingerprints. However, learned fingerprints require large training data sets, are inherently biased, and are based on complex deep learning architectures. Here we present the differential reaction fingerprint DRFP. The DRFP algorithm takes a reaction SMILES as an input and creates a binary fingerprint based on the symmetric difference of two sets containing the circular molecular n-grams generated from the molecules listed left and right from the reaction arrow, respectively, without the need for distinguishing between reactants and reagents. We show that DRFP performs better than DFT-based fingerprints in reaction yield prediction and other structure-based fingerprints in reaction classification, reaching the performance of state-of-the-art learned fingerprints in both tasks while being data-independent.

Synergistic effects of antimicrobial peptide dendrimer-chitosan polymer conjugates against Pseudomonas aeruginosa.

We report herein a new chemical platform for coupling chitosan derivatives to antimicrobial peptide dendrimers (AMPDs) with different degrees of ramification and molecular weights via thiol-maleimide reactions. Previous studies showed that simple incorporation of AMPDs to polymeric hydrogels resulted in a loss of antibacterial activity and augmented cytotoxicity to mammalian cells. We have shown that coupling AMPDs to chitosan derivatives enabled the two compounds to act synergistically. We showed that the antimicrobial activity was preserved when incorporating AMPD conjugates into various biopolymer formulations, including nanoparticles, gels, and foams. Investigating their mechanism of action using electron and time-lapse microscopy, we showed that the AMPD-chitosan conjugates were internalized after damaging outer and inner Gram-negative bacterial membranes. We also showed the absence of AMPD conjugates toxicity to mammalian cells. This chemical technological platform could be used for the development of new membrane disruptive therapeutics to eradicate pathogens present in acute and chronic wounds.