Ternary Complexes of BiI3/CuI and SbI3/CuI with Tetrahydrothiophene.

Reactions of BiI3/CuI mixtures with tetrahydrothiophene (THT) in toluene produce 2-D sheet networks BiCu3I6(THT)n (n = 2, 3, or 4), depending on reaction conditions. All three structures are based on BiI6 octahedra, which share pairs of (µ2-I)2 with Cu3(THT)n units. BiCu3I6(THT)2 features Cu2(µ2-I)2 rhombs with close Cu···Cu interactions and is accompanied by formation of the very complex HBi3Cu12I22(THT)8. Reactions of SbI3/CuI with THT in toluene produced a SbCu3I6(THT)2 network shows Cu3(µ2-THT)2 units, like its Bi congener, but Cu6(µ2-I)6 barrels rather than rhombs. Isolated SbI3 units are stacked above the Cu6I6 barrels. A molecular compound, Sb3Cu3I12(THT)6 consists of a face-sharing Sb3I12 stack, in which the Cu-THT units are bonded in asymmetric fashion about the central SbI6. Metal-halide bonds were investigated via QTAIM and NLMO analyses, demonstrating that these bonds are largely ionic and occur between the Bi/Sb and I p orbitals. Hirshfeld analysis shows significant H···H and H···I interactions. Diffuse reflectance spectroscopy (DRS) reveals band edges for the Bi species of 1.71-1.82 eV, while those for the neutral Sb complexes are in the range of 1.94-2.06 eV. Mapping of the electronic structure via density of state calculations indicates population of antibonding Bi/Sb-I orbitals in the excited state.

Molecular Interactions Required for Activation of Complement Component C2 Include Exosites Located on the Serine Protease Domain of C1s and Mannose-Binding Lectin Associated Protease-2.

The activation of the CP/LP C3 proconvertase complex is a key event in complement activation and involves cleavage of C4 and C2 by the C1s protease (classical pathway) or the mannose-binding lectin-associated serine protease (MASP)-2 (lectin pathway). Efficient cleavage of C4 by C1s and MASP-2 involves exosites on the complement control protein and serine protease (SP) domains of the proteases. The complement control protein domain exosite is not involved in cleavage of C2 by the proteases, but the role of an anion-binding exosite (ABE) on the SP domains of the proteases has (to our knowledge) never been investigated. In this study, we have shown that the ABE on the SP of both C1s and MASP-2 is crucial for efficient cleavage of C2, with mutant forms of the proteases greatly impaired in their rate of cleavage of C2. We have additionally shown that the site of binding for the ABE of the proteases is very likely to be located on the von Willebrand factor domain of C2, with the precise area differing between the enzymes: whereas C1s requires two anionic clusters on the von Willebrand factor domain to enact efficient cleavage of C2, MASP-2 apparently only requires one. These data provide (to our knowledge) new information about the molecular determinants for efficient activation of C2 by C1s and MASP-2. The enhanced view of the molecular events underlying the early stages of complement activation provides further possible intervention points for control of this activation that is involved in a number of inflammatory diseases.

Crystal structure and Hirshfeld surface analysis of cyclo-tetra-bromido-1κ2Br,3κ2Br-tetra-kis-(µ2-2-{[(pyridin-2-yl)meth-yl]amino}-ethane-1-thiol-ato-κ3N,S:S)tetra-mercury(II).

The macrometallacyclic title compound, [Hg4Br4(C8H11N2S)4] or [((HgL 2)(HgBr2))2] (1) where HL = 2-{[(pyridin-2-yl)meth-yl]amino}-ethane-1-thiol, was prepared and structurally characterized. The Hg2+ complex crystallizes in the P21/c space group. The centrosymmetric Hg4S4 metallacycle is constructed from metal ions with alternating distorted tetra-hedral Br2S2 and distorted seesaw N2S2 primary coordination environments with pendant pyridyl groups. The backfolded extended chair metallacycle conformation suggests inter-actions between each of the bis-chelated mercury atoms and Br atoms lying above and below the central Hg2S4 plane. Supra-molecular inter-actions in 1 include a fourfold aryl embrace and potential hydrogen bonds with bromine as the acceptor. Hirshfeld surface analysis indicates that H⋯H (51.7%), Br⋯H/H⋯Br (23.0%) and C⋯H/H⋯C (9.5%) inter-actions are dominant.

Rapid single crystal growth via guest displacement from host-guest complexes.

The preparation of single crystals of sparingly soluble polycyclic aromatic compounds in MeCN is facilitated by solubilizing ionic host-guest complexation under otherwise poor solvent conditions. The guest is then crystallized within minutes through controlled guest displacement from the host via competitor equilibria, or over days through direct crystallization of the host-guest complex itself.

Effect of a protease-activated receptor-2 antagonist (GB88) on inflammation-related loss of alveolar bone in periodontal disease.

BACKGROUND AND OBJECTIVE: Protease-activated receptor-2 (PAR2 ), a pro-inflammatory G-protein coupled receptor, has been associated with pathogenesis of periodontitis and the resulting bone loss caused by oral pathogens, including the keystone pathogen Porphyromonas gingivalis (P. gingivalis). We hypothesised that administration of a PAR2 antagonist, GB88, might prevent inflammation and subsequent alveolar bone resorption in a mouse model of periodontal disease. METHODS: Periodontitis was induced in mice by oral inoculations with P. gingivalis for a total of eight times over 24 days. The infected mice were treated with either GB88 or vehicle for the duration of the trial. Following euthanasia on day 56, serum was collected and used for the detection of mast cell tryptase. The right maxillae were defleshed and stained with methylene blue to measure the exposed cementum in molar teeth. The left maxillae were prepared for cryosections followed by staining for tartrate-resistant acid phosphatase to identify osteoclasts or with toluidine blue to identify mast cells. Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of inflammatory cytokines in the gingival tissue. Supernatants of T-lymphocyte cultures isolated from the regional lymph nodes were assayed using a cytometric bead array to measure the Th1/Th2/Th17 cytokine levels. RESULTS: Measurement of the exposed cementum showed that GB88 reduced P. gingivalis-induced alveolar bone loss by up to 69%. GB88 also prevented the increase in osteoclast numbers observed in the infected mice. Serum tryptase levels were significantly elevated in both the infected groups, and not altered by treatment. RT-qPCR showed that GB88 prevented the upregulation of Il1b, Il6, Ifng and Cd11b. In T-lymphocyte supernatants, only IFNγ and IL-17A levels were increased in response to infection, but this was prevented by GB88 treatment. CONCLUSIONS: GB88 significantly reduced osteoclastic alveolar bone loss in mice infected with P. gingivalis, seemingly by preventing the upregulation of several inflammatory cytokines. PAR2 antagonism may be an effective treatment strategy for periodontal disease.

Homo- and Heteronuclear Group 12 Metallothionein Type B Cluster Analogs: Synthesis, Structure, 1H NMR and ESI-MS.

Metallothioneins (MTs) are a ubiquitous class of small cysteine-rich metal-binding proteins involved in metal homeostasis and detoxification with highly versatile metal binding properties. Despite the long-standing association of MT with M3S3 and M4S5 metal clusters, synthetic complexes with these core architectures are exceptionally rare. Here, we demonstrate an approach to synthesizing and characterizing aggregates of group 12 metal ions with monocyclic M3S3 cores in acetonitrile solution without the protection of a protein. Multidentate monothiol ligand N,N-bis(2-pyridylmethyl)-2-aminoethanethiol (L1H) provided [Cd3(L1)3](ClO4)3 (1), the first structurally characterized nonproteinaceous aggregate with a metallothionein-like monocyclic Cd3S3 core. In addition, [Zn3(L1)3](ClO4)3·4CH3CN (2·4CH3CN) was characterized by X-ray crystallography. The complex cations of 1 and 2 had comparable structures despite being nonisomorphic. Variable temperature and concentration 1H NMR were used to investigate aggregation equilibria of 1, 2, and a precipitate with composition "Hg(L1)(ClO4)" (3). Cryogenic 1H NMR studies of 3 revealed a J(199Hg1H) coupling constant pattern consistent with an aggregate possessing a cyclic core. ESI-MS was used for gas-phase characterization of 1-3, as well as mixed-metal [M2M'(L1)3(ClO4)2]+ ions prepared in situ by pairwise acetonitrile solution combinations of the group 12 complexes of L1. Access to synthetic variants of metallothionein-like group 12 aggregates provides an additional approach to understanding their behavior.

Pyridine interaction with γ-CuI: synergy between molecular dynamics and molecular orbital approaches to molecule/surface interactions.

We have used a synergistic computational approach merging Molecular Dynamics (MD) simulations with density functional theory (DFT) to investigate the mechanistic aspects of chemisorption of pyridine (Py) molecules on copper iodide. The presence of both positive and negative ions at the metal halide surface presents a chemical environment in which pyridine molecules may act as charge donors and/or acceptors. Computational results reveal that Py molecules interact with the γ-CuI(111) surface owing to a combination of noncovalent Cu⋯N, Cu/I⋯π/π*, and hydrogen bonding interactions as determined via Natural Bonding Orbitals (NBO). Introduction of surface defect sites alters the interaction dynamics, resulting in a "localizing effect" in which the Py molecules clump together within the defect site. Significant enhancement of hydrogen bonding between C-H σ* and I 6p orbitals results in more tightly surface-bound Py molecules. Our findings provide a platform for understanding the interaction between Py and Py-derivative vapors and metal-based surfaces that contain both electron acceptor and donor atoms.

Mapping the binding site of C1-inhibitor for polyanion cofactors.

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.

Understanding the vapochromic response of mixed copper(i) iodide/silver(i) Iodide nanoparticles toward dimethyl sulfide.

We report on the vapochromic behavior of a series of homo- and heterometallic copper(i) iodide/silver(i) iodide nanoparticles when exposed to dimethyl sulfide (DMS) vapor. These systems show remarkable colorimetric sensing behavior via emission color upon DMS exposure, shifting from pink to green emission. Kinetics measurements of CuI/AgI nanoparticle reactions with DMS show a significant rate increase with increasing Ag(i) content. However, luminescence spectroscopy and X-ray diffraction of the post-exposure samples with varying Ag(i) content reveal that the luminophore is identical in all cases and contains no Ag(i) ions. To rationalize the experimental observations and determine the vapochromic response mechanism, molecular dynamic calculations were performed on model (111) cation-terminated surfaces of copper iodide crystals doped with variable amounts of silver. Computational studies indicate that heterometallic Cu/Ag systems have a stronger binding affinity towards DMS vapor molecules than homometallic CuI and that embedding of the DMS molecules into the surface is the primary intermediate by which the vapochromic response occurs.

Alkyl pyridinium iodocyanocuprate(i) chains (RPy)2[Cu2I3(CN)]: insight into structural, electronic and spectroscopic properties.

Equimolar mixtures of copper(i) iodide (CuI) and copper(i) cyanide (CuCN) react with N-alkyl pyridinium iodides (RPy+I-, R = Me, Et, n-propyl = Pr, and n-butyl = Bu) to produce pyridinium iodocyanocuprate(i) salts, (RPy)2[Cu2I3CN]. Crystal structures reveal isostructural anionic chains consisting of trigonal pyramidal Cu2(µ2-I)3 clusters bridged by C/N-disordered cyano units. The 1-D chains are nearly linear but vary with respect to whether adjacent clusters are staggered or eclipsed. A detailed investigation via Hirshfeld surface analysis reveals that hydrogen bonding between the triiodide group and pyridinium cation are the driver for assembly in these systems. Interestingly, spectroscopic investigations of absorption edge and emission energies show a general red shift with increasing hydrogen bonding. DFT and TD-DFT calculations were used to determine the electronic structure and band assignment of these materials to elucidate the nature behind this structure/function relationship.

Mild, Modular, and Convergent Synthesis of Helical Naphtho[2,1-c]chromenes via a Multistep Cyclization/Aromatization Cascade Sequence.

Tetracyclic 6H-naphtho[2,1-c]chromenes are expeditiously synthesized through a BF3·OEt2-mediated, three-step cascade reaction, creating new central pyran and aromatic rings. The cascade involves the addition of phenol-derived alkynyl substrates to BF3-activated aldehydes followed by alkyne-Prins cyclization, Friedel-Crafts reaction, and final elimination. Aliphatic and electron-deficient aromatic aldehydes afford the products in 50-74% isolated yields, but benzaldehyde and tolualdehyde resulted in lower yields. X-ray analysis of a p-bromophenyl derivative (5aA) shows the two aromatic moieties are twisted by 28° to create a helical backbone.

Protease-associated import systems are widespread in Gram-negative bacteria.

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.

Determination of the crystal structure and substrate specificity of ananain.

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M-1s-1, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E-64, solved at 1.73 and 1.98 Å, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E-64 and ananain explains the high inhibitory efficiency of E-64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.

Synthesis and Luminescence of Optical Memory Active Tetramethylammonium Cyanocuprate(I) 3D Networks.

The structures of three tetramethylammonium cyanocuprate(I) 3D networks [NMe4]2[Cu(CN)2]2•0.25H2O (1), [NMe4][Cu3(CN)4] (2), and [NMe4][Cu2(CN)3] (3), (Me4N = tetramethylammonium), and the photophysics of 1 and 2 are reported. These complexes are prepared by combining aqueous solutions of the simple salts tetramethylammonium chloride and potassium dicyanocuprate. Single-crystal X-ray diffraction analysis of complex 1 reveals {Cu2(CN)2(µ2-CN)4} rhomboids crosslinked by cyano ligands and D3h {Cu(CN)3} metal clusters into a 3D coordination polymer, while 2 features independent 2D layers of fused hexagonal {Cu8(CN)8} rings where two Cu(I) centers reside in a linear C∞v coordination sphere. Metallophilic interactions are observed in 1 as close Cu⋯Cu distances, but are noticeably absent in 2. Complex 3 is a simple honeycomb sheet composed of trigonal planar Cu(I) centers with no Cu…Cu interactions. Temperature and time-dependent luminescence of 1 and 2 have been performed between 298 K and 78 K and demonstrate that 1 is a dual singlet/triplet emitter at low temperatures while 2 is a triplet-only emitter. DFT and TD-DFT calculations were used to help interpret the experimental findings. Optical memory experiments show that 1 and 2 are both optical memory active. These complexes undergo a reduction of emission intensity upon laser irradiation at 255 nm although this loss is much faster in 2. The loss of emission intensity is reversible in both cases by applying heat to the sample. We propose a light-induced electron transfer mechanism for the optical memory behavior observed.

Twenty years of bioinformatics research for protease-specific substrate and cleavage site prediction: a comprehensive revisit and benchmarking of existing methods.

The roles of proteolytic cleavage have been intensively investigated and discussed during the past two decades. This irreversible chemical process has been frequently reported to influence a number of crucial biological processes (BPs), such as cell cycle, protein regulation and inflammation. A number of advanced studies have been published aiming at deciphering the mechanisms of proteolytic cleavage. Given its significance and the large number of functionally enriched substrates targeted by specific proteases, many computational approaches have been established for accurate prediction of protease-specific substrates and their cleavage sites. Consequently, there is an urgent need to systematically assess the state-of-the-art computational approaches for protease-specific cleavage site prediction to further advance the existing methodologies and to improve the prediction performance. With this goal in mind, in this article, we carefully evaluated a total of 19 computational methods (including 8 scoring function-based methods and 11 machine learning-based methods) in terms of their underlying algorithm, calculated features, performance evaluation and software usability. Then, extensive independent tests were performed to assess the robustness and scalability of the reviewed methods using our carefully prepared independent test data sets with 3641 cleavage sites (specific to 10 proteases). The comparative experimental results demonstrate that PROSPERous is the most accurate generic method for predicting eight protease-specific cleavage sites, while GPS-CCD and LabCaS outperformed other predictors for calpain-specific cleavage sites. Based on our review, we then outlined some potential ways to improve the prediction performance and ease the computational burden by applying ensemble learning, deep learning, positive unlabeled learning and parallel and distributed computing techniques. We anticipate that our study will serve as a practical and useful guide for interested readers to further advance next-generation bioinformatics tools for protease-specific cleavage site prediction.

Synthesis of Spirocyclic Diphosphite-Supported Gold Metallomacrocycles via a Protodeauration/Cyclization Strategy: Mechanistic and Binding Studies.

A spirocylic diphosphite was used to generate P-metalated bimetallic complexes through protodeauration reactions involving LAuC6H4tBu (L = JohnPhos, tBuXPhos) and metallomacrocycles through protodeauration/cyclization using tBuC6H4AuP^PAuC6H4tBu precursors (P^P = flexible diphosphine). While the synthesis of the bimetallic complexes followed a stepwise process, generation of the metallomacrocycles was highly complex because of a series of reversible ligand redistribution reactions. The self-assembly was monitored, and key intermediates were identified by NMR spectroscopy and high-resolution mass spectrometry. The mechanistic investigation showed that using flexible diphosphine linkers was critical to the selective synthesis of metallomacrocycles because rigid diphosphines generated intractable mixtures of linear and cyclic compounds. The X-ray structure of a 32-membered metallomacrocycle revealed that the compound crystallized in an unsymmetrical collapsed form that was held together by two supported aurophilic interactions while the flexible diphosphines were folded along opposite sides of the metallomacrocycle. The solution structure was consistent with a symmetric species, which suggested interconversion between an open and collapsed form and/or rapid twisting of a collapsed form. The 32-membered metallomacrocycle was used to bind estrogen primarily through the formation of AuP-O-···H-OR hydrogen bonds.

Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease.

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1ß, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.

Molecular basis for the folding of ß-helical autotransporter passenger domains.

Bacterial autotransporters comprise a C-terminal ß-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated ß-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter ß-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the ß-hairpin structure of the fifth extracellular loop of the ß-barrel domain has a crucial role for passenger domain folding into a ß-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with ß-helical passenger domains. We propose that the autotransporter ß-barrel domain is a folding vector that nucleates folding of the passenger domain.

Alkyl Pyridinium Iodocuprate(I) Clusters: Structural Types and Charge Transfer Behavior.

The reaction of copper(I) iodide (CuI) and N-alkyl pyridinium (RPy+, R = H, Me, Et, n-propyl = Pr, n-butyl = Bu, n-pentyl = Pn, and n-hexyl = Hx) or N-butyl-3-substituted pyridinium (N-Bu-3-PyX+, X = I, Br, Cl, CN, and OMe) iodide salts yielded pyridinium iodocuprate(I) salts. Crystal structures of iodocuprate ions coupled with RPy+ include {Cu3I6 3-} n (R = H), {Cu2I3 -} n (R = Me), {Cu3I4 -} n (R = Et), {Cu6I8 2-} n (R = Pr), and {Cu5I7 2-} n (R = Bu, Pn, Hx). The [N-Bu-3-PyX]+ ions were typically paired with the 1-D chain {Cu5I7 2-} n . Diffuse reflectance spectroscopy performed on the [N-Bu-3-PyX]+ iodocuprate salts revealed that increasing the electron withdrawing capacity of the [N-Bu-3-PyX]+ system reduced the absorption edge of the iodocuprate salt. Variable temperature emission spectra of several [N-Bu-3-PyX]+ compounds revealed two emission peaks, one consistent with a cluster-centered halide to metal charge transfer and the other consistent with an intermolecular mixed halide/metal charge transfer to the organic cation. The emission intensity and emission wavelength of the mixed halide/metal to cation charge transfer depends on the organic cation substitution.

PROSPERous: high-throughput prediction of substrate cleavage sites for 90 proteases with improved accuracy.

Summary: Proteases are enzymes that specifically cleave the peptide backbone of their target proteins. As an important type of irreversible post-translational modification, protein cleavage underlies many key physiological processes. When dysregulated, proteases' actions are associated with numerous diseases. Many proteases are highly specific, cleaving only those target substrates that present certain particular amino acid sequence patterns. Therefore, tools that successfully identify potential target substrates for proteases may also identify previously unknown, physiologically relevant cleavage sites, thus providing insights into biological processes and guiding hypothesis-driven experiments aimed at verifying protease-substrate interaction. In this work, we present PROSPERous, a tool for rapid in silico prediction of protease-specific cleavage sites in substrate sequences. Our tool is based on logistic regression models and uses different scoring functions and their pairwise combinations to subsequently predict potential cleavage sites. PROSPERous represents a state-of-the-art tool that enables fast, accurate and high-throughput prediction of substrate cleavage sites for 90 proteases. Availability and implementation: http://prosperous.erc.monash.edu/. Contact: jiangning.song@monash.edu or geoff.webb@monash.edu or r.pike@latrobe.edu.au. Supplementary information: Supplementary data are available at Bioinformatics online.