Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

A Compact Reprogrammed Genetic Code for De Novo Discovery of Proteolytically Stable Thiopeptides.

Thiopeptides make up a group of structurally complex peptidic natural products holding promise in bioengineering applications. The previously established thiopeptide/mRNA display platform enables de novo discovery of natural product-like thiopeptides with designed bioactivities. However, in contrast to natural thiopeptides, the discovered structures are composed predominantly of proteinogenic amino acids, which results in low metabolic stability in many cases. Here, we redevelop the platform and demonstrate that the utilization of compact reprogrammed genetic codes in mRNA display libraries can lead to the discovery of thiopeptides predominantly composed of nonproteinogenic structural elements. We demonstrate the feasibility of our designs by conducting affinity selections against Traf2- and NCK-interacting kinase (TNIK). The experiment identified a series of thiopeptides with high affinity to the target protein (the best KD = 2.1 nM) and kinase inhibitory activity (the best IC50 = 0.15 µM). The discovered compounds, which bore as many as 15 nonproteinogenic amino acids in an 18-residue macrocycle, demonstrated high metabolic stability in human serum with a half-life of up to 99 h. An X-ray cocrystal structure of TNIK in complex with a discovered thiopeptide revealed how nonproteinogenic building blocks facilitate the target engagement and orchestrate the folding of the thiopeptide into a noncanonical conformation. Altogether, the established platform takes a step toward the discovery of thiopeptides with high metabolic stability for early drug discovery applications.

Deep Learning-Driven Library Design for the De Novo Discovery of Bioactive Thiopeptides.

Broad substrate tolerance of ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic enzymes has allowed numerous strategies for RiPP engineering. However, despite relaxed specificities, exact substrate preferences of RiPP enzymes are often difficult to pinpoint. Thus, when designing combinatorial libraries of RiPP precursors, balancing the compound diversity with the substrate fitness can be challenging. Here, we employed a deep learning model to streamline the design of mRNA display libraries. Using an in vitro reconstituted thiopeptide biosynthesis platform, we performed mRNA display-based profiling of substrate fitness for the biosynthetic pathway involving five enzymes to train an accurate deep learning model. We then utilized the model to design optimal mRNA libraries and demonstrated their utility in affinity selections against IRAK4 kinase and the TLR10 cell surface receptor. The selections led to the discovery of potent thiopeptide ligands against both target proteins (KD up to 1.3 nM for the best compound against IRAK4 and 300 nM for TLR10). The IRAK4-targeting compounds also inhibited the kinase at single-digit µM concentrations in vitro, exhibited efficient internalization into HEK293H cells, and suppressed NF-kB-mediated signaling in cells. Altogether, the developed approach streamlines the discovery of pseudonatural RiPPs with de novo designed biological activities and favorable pharmacological properties.

Coordination Polymers of Polyphenyl-Substituted Potassium Cyclopentadienides.

A series of potassium salts of di- and tri-arylsubstituted cyclopentadienes has been obtained by the metalation of the corresponding cyclopentadienes with benzylpotassium in THF media. Crystals of all compounds, afforded by recrystallization from THF/hexane, diglyme-THF/hexane and toluene/hexane mixtures, have been studied by X-ray diffraction. All studied potassium cyclopentadienides exhibit the luminescence at room temperature and overall quantum yield of photoluminescence for potassium salt of diarylsubstituted cyclopentadiene is 18%.

Solid-Phase-Based Synthesis of Lactazole-Like Thiopeptides.

A strategy for the synthesis of de novo discovered lactazole-like thiopeptides is reported. The approach revolves around a convergent and scalable preparation of the central triheterocyclic amino acid and its utilization in Fmoc solid-phase peptide synthesis for modular peptide chain assembly. A technique for preparing C-terminally functionalized thiopeptides for biological studies is also described. The syntheses of 11 TNIK-inhibitor thiopeptides and 6 of their derivatives in multimilligram quantities highlight the practical utility of the developed protocols.

De Novo Discovery of Thiopeptide Pseudo-natural Products Acting as Potent and Selective TNIK Kinase Inhibitors.

Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥1012) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affinity selection against Traf2- and NCK-interacting kinase (TNIK), a protein involved in several cancers, which yielded a plethora of candidate thiopeptides. Of the 11 synthesized compounds, 9 had high affinities for the target kinase (best KD = 1.2 nM) and 10 inhibited its enzymatic activity (best Ki = 3 nM). X-ray structural analysis of the TNIK/thiopeptide interaction revealed the unique mode of substrate-competitive inhibition exhibited by two of the discovered compounds. The thiopeptides internalized to the cytosol of HEK293H cells as efficiently as the known cell-penetrating peptide Tat (4-6 µM). Accordingly, the most potent compound, TP15, inhibited TNIK in HCT116 cells. Altogether, our platform enables the exploration of pseudo-natural thiopeptides with favorable pharmacological properties in drug discovery applications.

Accurate Models of Substrate Preferences of Post-Translational Modification Enzymes from a Combination of mRNA Display and Deep Learning.

Promiscuous post-translational modification (PTM) enzymes often display nonobvious substrate preferences by acting on diverse yet well-defined sets of peptides and/or proteins. Understanding of substrate fitness landscapes for PTM enzymes is important in many areas of contemporary science, including natural product biosynthesis, molecular biology, and biotechnology. Here, we report an integrated platform for accurate profiling of substrate preferences for PTM enzymes. The platform features (i) a combination of mRNA display with next-generation sequencing as an ultrahigh throughput technique for data acquisition and (ii) deep learning for data analysis. The high accuracy (>0.99 in each of two studies) of the resulting deep learning models enables comprehensive analysis of enzymatic substrate preferences. The models can quantify fitness across sequence space, map modification sites, and identify important amino acids in the substrate. To benchmark the platform, we performed profiling of a Ser dehydratase (LazBF) and a Cys/Ser cyclodehydratase (LazDEF), two enzymes from the lactazole biosynthesis pathway. In both studies, our results point to complex enzymatic preferences, which, particularly for LazBF, cannot be reduced to a set of simple rules. The ability of the constructed models to dissect such complexity suggests that the developed platform can facilitate a wider study of PTM enzymes.

Site-Specific Nonenzymatic Peptide S/O-Glutamylation Reveals the Extent of Substrate Promiscuity in Glutamate Elimination Domains.

Formation of dehydroalanine and dehydrobutyrine residues via tRNA-dependent dehydration of serine and threonine is a key post-translational modification in the biosynthesis of lanthipeptide and thiopeptide RiPPs. The dehydration process involves two reactions, wherein the O-glutamyl Ser/Thr intermediate, accessed by a dedicated enzyme utilizing Glu-tRNAGlu as the acyl donor, is recognized by the second enzyme, referred to as the glutamate elimination domain (ED), which catalyzes the eponymous reaction yielding a dehydroamino acid. Many details of ED catalysis remain unexplored because the scope of available substrates for testing is limited to those that the upstream enzymes can furnish. Here, we report two complementary strategies for direct, nonenzymatic access to diverse ED substrates. We establish that a thiol-thioester exchange reaction between a Cys-containing peptide and an α thioester of glutamic acid leads an S-glutamylated intermediate which can act as a substrate for EDs. Furthermore, we show that the native O-glutamylated substrates can be accessible from S-glutamylated peptides upon a site-specific S-to-O acyl transfer reaction. Combined with flexible in vitro translation utilized for rapid peptide production, these chemistries enabled us to dissect the substrate recognition requirements of three known EDs. Our results establish that EDs are uniquely promiscuous enzymes capable of acting on substrates with arbitrary amino acid sequences and performing retro-Michael reaction beyond the canonical glutamate elimination. To facilitate substrate recruitment, EDs apparently engage in nonspecific hydrophobic interactions with their substrates. Altogether, our results establish the substrate scope of EDs and provide clues to their catalysis.

Accurate Broadcasting of Substrate Fitness for Lactazole Biosynthetic Pathway from Reactivity-Profiling mRNA Display.

We report a method for the high-throughput reactivity profiling of genetically encoded libraries as a tool to study substrate fitness landscapes for RiPP (ribosomally synthesized and post-translationally modified peptide) biosynthetic enzymes. This method allowed us to rapidly analyze the substrate preferences of the lactazole biosynthetic pathway using a saturation mutagenesis mRNA display library of lactazole precursor peptides. We demonstrate that the assay produces accurate and reproducible in vitro data, enabling the quantification of reaction yields with temporal resolution. Our results recapitulate the previously established knowledge on lactazole biosynthesis and expand it by identifying the extent of substrate promiscuity exhibited by the enzymes. This work lays a foundation for the construction and screening of mRNA display-based combinatorial thiopeptide libraries for the discovery of lactazole-inspired thiopeptides with de novo designed biological activities.

Raman Spectroscopy Study of Structurally Uniform Hydrogenated Oligomers of α-Olefins.

The expansion of the range of physico-chemical methods in the study of industrially significant α-olefin oligomers and polymers is of particular interest. In our article, we present a comparative Raman study of structurally uniform hydrogenated dimers, trimers, tetramers, and pentamers of 1-hexene and 1-octene, that are attractive as bases for freeze-resistant engine oils and lubricants. We found out that the joint monitoring of the disorder longitudinal acoustic mode (D-LAM) and symmetric C-C stretching modes allows the quantitative characterization of the number and length of alkyl chains (i.e., two structural characteristics), upon which the pour point and viscosity of the hydrocarbons depend, and to distinguish these compounds from both each other and linear alkanes. We demonstrated that the ratio of the contents of CH2 and CH3 groups in these hydrocarbons can be determined by using the intensities of the bands in the spectra, related to the asymmetric stretching vibrations of these groups. The density functional theory (DFT) calculations were applied to reveal the relations between the wavenumber and bandshape of the symmetric C-C stretching mode and a conformation arrangement of the 1-hexene and 1-octene dimers. We found that the branched double-chain conformation results in the splitting of the C-C mode into two components with the wavenumbers, which can be used as a measure of the length of branches. This conformation is preferable to the extended-chain conformation for hydrogenated 1-hexene and 1-octene dimers.

Introduction to Thiopeptides: Biological Activity, Biosynthesis, and Strategies for Functional Reprogramming.

Thiopeptides (also known as thiazolyl peptides) are structurally complex natural products with rich biological activities. Known for over 70 years for potent killing of Gram-positive bacteria, thiopeptides are experiencing a resurgence of interest in the last decade, primarily brought about by the genomic revolution of the 21st century. Every area of thiopeptide research-from elucidating their biological function and biosynthesis to expanding their structural diversity through genome mining-has made great strides in recent years. These advances lay the foundation for and inspire novel strategies for thiopeptide engineering. Accordingly, a number of diverse approaches are being actively pursued in the hope of developing the next generation of natural-product-inspired therapeutics. Here, we review the contemporary understanding of thiopeptide biological activities, biosynthetic pathways, and approaches to structural and functional reprogramming, with a special focus on the latter.

Promiscuous Enzymes Cooperate at the Substrate Level En Route to Lactazole A.

Enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, yet usually the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate the biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level and supplement this study with substrate scope analysis for participating enzymes. Our results reveal an unusual but well-defined assembly process where cyclodehydration, dehydroalanine formation, and azoline dehydrogenation events are intertwined due to minimal substrate recognition requirements characteristic of every lactazole enzyme. Additionally, each enzyme plays a role in directing LazBF-mediated dehydroalanine formation, which emerges as the central theme of the assembly process. Cyclodehydratase LazDE discriminates a single serine residue for azoline formation, leaving the remaining five as potential dehydratase substrates. Pyridine synthase LazC exerts kinetic control over LazBF to prevent the formation of overdehydrated thiopeptides, whereas the coupling of dehydrogenation to dehydroalanine installation impedes generation of underdehydrated products. Altogether, our results indicate that substrate-level cooperation between the biosynthetic enzymes maintains the integrity of lactazole assembly. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.

Minimal lactazole scaffold for in vitro thiopeptide bioengineering.

Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we establish a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis reveals remarkable substrate tolerance of the biosynthetic enzymes and leads to the development of the minimal lactazole scaffold, a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz opens access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and 18 amino acid-long tail regions, as well as to hybrid thiopeptides containing non-proteinogenic amino acids. This work suggests that the minimal lactazole scaffold is amenable to extensive bioengineering and opens possibilities to explore untapped chemical space of thiopeptides.

FTIR-based L-asparaginase activity assay enables continuous measurements in optically dense media including blood plasma.

Complex heterogeneous systems, such as micelles or blood plasma, represent a particularly challenging environment to measure the catalytic parameters of some enzymes, including l-asparaginase. Existing methods are strongly interfered by the presence of plasma proteins, amino acids, as well as other components of plasma. Here we show that FTIR spectroscopy enables continuous real-time measurement of catalytic activity of l-asparaginase, in native and in PEG-chitosan conjugated form, in aqueous solutions as well as in heterogeneous non-transparent multicomponent systems, including colloidal systems or blood plasma, with minimal or no sample preparation. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not allow direct measurement with absorption or fluorescence spectroscopy.

Crystal structure of bis(µ2-tri-phenyl-acetato-κO:κO')bis(diisobutylaluminium).

Single crystals of the title compound, [Al(iBu)2(O2CCPh3)]2 or [Al2(C4H9)4(C20H15O2)2], have been formed in the reaction between tris-(tetra-hydro-furan)-tris-(tri-phenyl-acetato)-neodymium, [Nd(Ph3CCOO)3(THF)3], and triiso-butyl-aluminium, Al(iBu)3, in hexane followed by low-temperature crystallization (243 K) from the reaction mixture. The structure has triclinic (P ) symmetry at 120 K. The dimeric complex [Al(iBu)2(O2CCPh3-µ-κO:κO')]2 is located about an inversion centre. The tri-phenyl-acetate ligand displays a µ-κO:κO'-bridging coordination mode, leading to the formation of an octa-gonal Al2O4C2 core. The complex displays HPh⋯CPh inter-molecular inter-actions.

Macrocyclic Peptides as Drug Candidates: Recent Progress and Remaining Challenges.

Peptides as a therapeutic modality attract much attention due to their synthetic accessibility, high degree of specific binding, and the ability to target protein surfaces traditionally considered "undruggable". Unfortunately, at the same time, other pharmacological properties of a generic peptide, such as metabolic stability and cell permeability, are quite poor, which limits the success of de novo discovered biologically active peptides as drug candidates. Here, we review how macrocyclization as well as the incorporation of nonproteogenic amino acids and various conjugation strategies may be utilized to improve on these characteristics to create better drug candidates. We analyze recent progress and remaining challenges in improving individual pharmacological properties of bioactive peptides, and offer our opinion on interfacing these, often conflicting, considerations, to create balanced drug candidates as a potential way to make further progress in this area.

Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides.

Thiopeptides are natural antibiotics that are fashioned from short peptides by multiple layers of post-translational modification. Their biosynthesis, in particular the pyridine synthases that form the macrocyclic antibiotic core, has attracted intensive research but is complicated by the challenges of reconstituting multiple-pathway enzymes. By combining select RiPP enzymes with cell free expression and flexizyme-based codon reprogramming, we have developed a benchtop biosynthesis of thiopeptide scaffolds. This strategy side-steps several challenges related to the investigation of thiopeptide enzymes and allows access to analytical quantities of new thiopeptide analogs. We further demonstrate that this strategy can be used to validate the activity of new pyridine synthases without the need to reconstitute the cognate prior pathway enzymes.

The structural diversity of heterocycle-fused potassium cyclopentadienides.

Cyclopentadienides of d- and f-elements are highly important complexes with undoubted potential for practical applications. Annelation of a heterocyclic fragment with an η5-ring results in substantial improvement of the catalytic properties of these compounds, called "heterocenes"; the investigation of metal coordination with these specific ligands is a highly important problem. We prepared potassium derivatives 5-8 of heterocycle-annelated cyclopentadienes with different structures - derivatives of cyclopenta[1,2-b:4,3-b']dithiophene (1), indeno[2,1-b]indole (2), indeno[1,2-b]indole (3), and indeno[1,2-b]indolizine (4) and studied the crystal and molecular structures of these salts by X-ray diffraction. We found that heterocycle-fused cyclopentadienides demonstrate remarkable diversity in metal-ligand coordination modes and crystal packing, with formation of two-dimensional polymeric (5), linear polymeric (6), tetrameric (7) and monomeric (8) structures. The NMR spectral data and results of DFT modeling indicate an increase in electron density in the cyclopentadienyl fragment, and this effect was found to be larger in the derivative of the new indolizine ligand precursor 4. The results of our study will be used in the design of next-generation catalysts of α-olefin polymerization.

Crystal structure of bis-(µ2-meth-anolato-κO:κO)hexa-methyl-bis-(µ2-tri-phenyl-acetato-κO:κO')bis-(µ2-tri-phenyl-acetato-κ2 O,O':κO)dialuminiumdi-lanthanum toluene tetra-solvate.

The title compound, [Al2La2(C20H15O2)4(CH3)6(CH3O)2]·4CH3C6H5 or [{La(Ph3CCOO)2(Me3AlOMe)}2]·4CH3C6H5, was formed in a reaction between lanthanum tris-(tetra-methyl-aluminate) and tri-phenyl-acetic acid (1:1) with unintended partial oxidation. The tri-phenyl-acetate ligand exhibits µ2-κ1 O:κ1 O' bridging and µ2-κ2 O,O':κ1 O semi-bridging coordination modes, forming a dimeric La2(µ-OCO)4 core. The semi-bridging tri-phenyl-acetate group provides additional bonding with an La3+ cation via the π-system of one of its phenyl rings. The tri-methyl-meth-oxy-aluminate anion, which is coordinated to the La3+ cation by its O atom, displays a rather long La-CMe bond. Two toluene mol-ecules are each disordered over two orientations about centres of symmetry with site occupancy factors of 0.5. The title compound represents the first example of an LnIII complex containing both alkyl alkoxide aluminate and π-bounded arene fragments.

Polyphenylcyclopentadienyl Ligands as an Effective Light-Harvesting π-Bonded Antenna for Lanthanide +3 Ions.

A new approach to design "antenna-ligands" to enhance the photoluminescence of lanthanide coordination compounds has been developed based on a π-type ligand-the polyphenyl-substituted cyclopentadienyl. The complexes of di-, tri-, and tetraphenyl cyclopentadienyl ligands with Tb and Gd have been synthesized and all the possible structural types from mononuclear to di- and tetranuclear complexes, as well as a coordination polymer were obtained. All types of the complexes have been studied by single-crystal X-ray diffraction and optical spectroscopy. All terbium complexes are luminescent at ambient temperature and two of them have relatively high quantum yields (50 and 60%). Analysis of energy transfer process has been performed and supported by quantum chemical calculations. The role of a low-lying intraligand charge transfer state formed by extra coordination with K+ in the Tb3+ ion luminescence sensitization is discussed. New aspects for design of lanthanide complexes containing π-type ligands with desired luminescence properties have been proposed.