Unique Cysteine Research Articles

Identification of a dual JAK3/TEC family kinase inhibitor for atopic dermatitis therapy.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by recurrent eczematous lesions and severe itching, for which clinical treatments are limited. Selectively inhibiting Janus Kinase 3 (JAK3) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) family kinases is proposed as a promising strategy to treat AD with possible reduced side effects and enhanced efficacy. In this study, we developed a dual JAK3/TEC family kinase inhibitor ZZB, which demonstrated potent inhibitory activity with IC50 values of 0.89nM against JAK3 and 11.56nM against TEC kinase interleukin-2-inducible T-cell kinase (ITK). Docking studies revealed that ZZB forms a covalent bond with the unique cysteine residue at position 909 (Cys909) in JAK3 and Cys442 in ITK. Utilizing human peripheral blood mononuclear cells, we discovered ZZB selectively inhibits JAK3-dependent cytokines signaling and ITK-mediated CD4+ T cell activation. Moreover, in vitro studies indicated ZZB significantly suppresses the proliferation and differentiation of CD4+ T cells, as well as the cytolytic function of CD8+ T cells and NK cells. We then conducted a pharmacokinetic study in mice and observed a favorable pharmacokinetic profile for ZZB. In a mouse model of AD induced by repeated application of 2,4-dinitrochlorobenzene to the shaved dorsal skin, oral administration of ZZB (100mg/kg) markedly improved skin condition and reduced immune cell infiltration, matching the efficacy of the positive drug dexamethasone. We conclude that the JAK3/TEC kinase inhibitor ZZB is a highly promising candidate for the treatment of AD.

Targeting PfCLK3 with Covalent Inhibitors: A Novel Strategy for Malaria Treatment.

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase PfCLK3 with the reversible inhibitor TCMDC-135051 (1), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

Effect of protease reaction conditions on volatile compounds generated in Maillard reaction products from soy protein hydrolysates

Maillard reaction products (MRPs) produced by heating protease-catalyzed soy protein hydrolysates (SPHs) with cysteine and ribose can generate meat-like flavors. However, the impact of protease reaction conditions on the volatile compound composition of MRPs has not been thoroughly investigated. In this study, seven SPHs were prepared using two proteases, flavourzyme and trypsin, over reaction times of 10, 120, and 1440 min. The volatile compound compositions, including sulfur-containing compounds, aldehydes, pyrazines, and furans, of the seven SPHs and the corresponding seven MRPs varied according to the protease reaction conditions and the Maillard reaction. Differences in pH, free amino acid composition, and peptide composition were responsible for these variations. Notably, soy-derived peptides containing unique cysteine sequences, such as PGCPST, DETICT, ECQIQK, and HCQR, were significantly reduced during the Maillard reaction, suggesting that these sequences may serve as precursors to volatile compounds.

Identification of Pathogenic Missense Mutations of NF1 Using Computational Approaches

Neurofibromatosis type 1 (NF1) is a prevalent autosomal dominant disorder caused by mutations in the NF1 gene, leading to multisystem disorders. Given the critical role of cysteine residues in protein stability and function, we aimed to identify key NF1 mutations affecting cysteine residues that significantly contribute to neurofibromatosis pathology. To identify the most critical mutations in the NF1 gene that contribute to the pathology of neurofibromatosis, we employed a sophisticated computational pipeline specifically designed to detect significant mutations affecting the NF1 gene. Our approach involved an exhaustive search of databases such as the Human Gene Mutation Database (HGMD), UniProt, and ClinVar for information on missense mutations associated with NF1. Our search yielded a total of 204 unique cysteine missense mutations. We then employed in silico prediction tools, including PredictSNP, iStable, and Align GVGD, to assess the impact of these mutations. Among the mutations, C379R, R1000C, and C1016Y stood out due to their deleterious effects on the biophysical properties of the neurofibromin protein, significantly destabilizing its structure. These mutations were subjected to further phenotyping analysis using SNPeffect 4.0, which predicted disturbances in the protein’s chaperone binding sites and overall structural stability. Furthermore, to directly visualize the impact of these mutations on protein structure, we utilized AlphaFold3 to simulate both the wild-type and mutant NF1 structures, revealing the significant effects of the R1000C mutation on the protein’s conformation. In conclusion, the identification of these mutations can play a pivotal role in advancing the field of precision medicine and aid in the development of effective drugs for associated diseases.

Mining unique cysteine synthetases and computational study on thoroughly eliminating feedback inhibition through tunnel engineering.

L-cysteine is an essential component in pharmaceutical and agricultural industries, and synthetic biology has made strides in developing new metabolic pathways for its production, particularly in archaea with unique O-phosphoserine sulfhydrylases (OPSS) as key enzymes. In this study, we employed database mining to identify a highly catalytic activity OPSS from Acetobacterium sp. (AsOPSS). However, it was observed that the enzymatic activity of AsOPSS suffered significant feedback inhibition from the product L-cysteine, exhibiting an IC50 value of merely 1.2 mM. A semi-rational design combined with tunnel analysis strategy was conducted to engineer AsOPSS. The best variant, AsOPSSA218R was achieved, totally eliminating product inhibition without sacrificing catalytic efficiency. Molecular docking and molecular dynamic simulations indicated that the binding conformation of AsOPSSA218R with L-cys was altered, leading to a reduced affinity between L-cysteine and the active pocket. Tunnel analysis revealed that the AsOPSSA218R variant reshaped the landscape of the tunnel, resulting in the construction of a new tunnel. Furthermore, random acceleration molecular dynamics simulation and umbrella sampling simulation demonstrated that the novel tunnel improved the suitability for product release and effectively separated the interference between the product release and substrate binding processes. Finally, more than 45 mM of L-cysteine was produced in vitro within 2 h using the AsOPSSA218R variant. Our findings emphasize the potential for relieving feedback inhibition by artificially generating new product release channels, while also laying an enzymatic foundation for efficient L-cysteine production.

An in silico design method of a peptide bioreceptor for cortisol using molecular modelling techniques

Cortisol is established as a reliable biomarker for stress prompting intensified research in developing wearable sensors to detect it via eccrine sweat. Since cortisol is present in sweat in trace quantities, typically 8–140 ng/mL, developing such biosensors necessitates the design of bioreceptors with appropriate sensitivity and selectivity. In this work, we present a systematic biomimetic methodology and a semi-automated high-throughput screening tool which enables rapid selection of bioreceptors as compared to ab initio design of peptides via computational peptidology. Candidate proteins from databases are selected via molecular docking and ranked according to their binding affinities by conducting automated AutoDock Vina scoring simulations. These candidate proteins are then validated via full atomistic steered molecular dynamics computations including umbrella sampling to estimate the potential of mean force using GROMACS version 2022.6. These explicit molecular dynamic calculations are carried out in an eccrine sweat environment taking into consideration the protein dynamics and solvent effects. Subsequently, we present a candidate baseline peptide bioreceptor selected as a contiguous sequence of amino acids from the selected protein binding pocket favourably interacting with the target ligand (i.e., cortisol) from the active binding site of the proteins and maintaining its tertiary structure. A unique cysteine residue introduced at the N-terminus allows orientation-specific surface immobilization of the peptide onto the gold electrodes and to ensure exposure of the binding site. Comparative binding affinity simulations of this peptide with the target ligand along with commonly interfering species e.g., progesterone, testosterone and glucose are also presented to demonstrate the validity of this proposed peptide as a candidate baseline bioreceptor for future cortisol biosensor development.

Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound (R,R,S)-26 has a kinact/KI of 4600 M-1 s-1, shows <1 μM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

Exploring the unmapped cysteine redox proteoform landscape.

Cysteine redox proteoforms define the diverse molecular states that proteins with cysteine residues can adopt. A protein with one cysteine residue must adopt one of two binary proteoforms: reduced or oxidized. Their numbers scale: a protein with 10 cysteine residues must assume one of 1,024 proteoforms. Although they play pivotal biological roles, the vast cysteine redox proteoform landscape comprising vast numbers of theoretical proteoforms remains largely uncharted. Progress is hampered by a general underappreciation of cysteine redox proteoforms, their intricate complexity, and the formidable challenges that they pose to existing methods. The present review advances cysteine redox proteoform theory, scrutinizes methodological barriers, and elaborates innovative technologies for detecting unique residue-defined cysteine redox proteoforms. For example, chemistry-enabled hybrid approaches combining the strengths of top-down mass spectrometry (TD-MS) and bottom-up mass spectrometry (BU-MS) for systematically cataloguing cysteine redox proteoforms are delineated. These methods provide the technological means to map uncharted redox terrain. To unravel hidden redox regulatory mechanisms, discover new biomarkers, and pinpoint therapeutic targets by mining the theoretical cysteine redox proteoform space, a community-wide initiative termed the "Human Cysteine Redox Proteoform Project" is proposed. Exploring the cysteine redox proteoform landscape could transform current understanding of redox biology.

Evaluation of a novel 177Lu-labelled therapeutic Affibody molecule with a deimmunized ABD domain and improved biodistribution profile

PurposeFusion of Affibody molecules with an albumin-binding domain (ABD) provides targeting agents, which are suitable for radionuclide therapy. To facilitate clinical translation, the low immunogenic potential of such constructs with targeting properties conserved is required.MethodsThe HER2-targeting Affibody molecule ZHER2:2891 was fused with a deimmunized ABD variant and DOTA was conjugated to a unique C-terminal cysteine. The novel construct, PEP49989, was labelled with 177Lu. Affinity, specificity, and in vivo targeting properties of [177Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated.ResultsThe maximum molar activity of 52 GBq/µmol [177Lu]Lu-PEP49989 was obtained. [177Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [177Lu]Lu-PEP49989 was similar to the affinity of [177Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [177Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [177Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [177Lu]Lu-PEP49989 (> 90 days) was significantly longer than the survival of mice treated with vehicle (38 days) or trastuzumab (45 days). Treatment using a combination of [177Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild.ConclusionIn preclinical studies, [177Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

Remodeling tumor-associated macrophage for anti-cancer effects by rational design of irreversible inhibition of mitogen-activated protein kinase-activated protein kinase 2.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) emerges as a pivotal target in developing anti-cancer therapies. The limitations of ATP-competitive inhibitors, due to insufficient potency and selectivity, underscore the urgent need for a covalent irreversible MK2 inhibitor. Our initial analyses of The Cancer Genome Atlas database revealed MK2's overexpression across various cancer types, especially those characterized by inflammation, linking it to poor prognosis and highlighting its significance. Investigating MK2's kinase domain led to the identification of a unique cysteine residue, enabling the creation of targeted covalent inhibitors. Compound 11 was developed, demonstrating robust MK2 inhibition (IC50=2.3nM) and high selectivity. It binds irreversibly to MK2, achieving prolonged signal suppression and reducing pathological inflammatory cytokines in macrophages. Furthermore, compound 11 or MK2 knockdown can inhibit the tumor-promoting macrophage M2 phenotype in vitro and in vivo. In macrophage-rich tumor model, compound 11 notably slowed growth in a dose-dependent manner. These findings support MK2 as a promising anticancer target, especially relevant in cancers fueled by inflammation or dominated by macrophages, and provide compound 11 serving as an invaluable chemical tool for exploring MK2's functions.

Design of a gold nanoparticles site in an engineered lipase: an artificial metalloenzyme with enantioselective reductase-like activity.

The conjugation of gold complexes with proteins has proved to be interesting and effective in obtaining artificial metalloenzymes as catalysts with improved properties such as higher stability, activity and selectivity. However, the design and precise regulation of their structure as protein nanostructured forms level remains a challenge. Here, we have designed and constructed a gold nanoparticles-enzyme bioconjugate, by tailoring the in situ formation of gold nanoparticles (AuNPs) at two specific sites on the structure of an alkalophilic lipase from Geobacillus thermocatenulatus (GTL). For this purpose, two genetically modified variants of GTL were created by inserting a unique cysteine residue into the catalytic active site by replacing the active serine (GTL-114) and into the lid site (GTL-193). The enzyme, after a first protein-gold coordination, induced the in situ formation of AuNPs, generating a homogeneous artificial enzyme. The size and morphology of the nanoparticles in the AuNPs-enzyme conjugate have been controlled by specific pH conditions in synthesis and the specific protein region where they are formed. Reductase activity of all of them was confirmed in the hydrogenation of nitroarenes in aqueous media. The protein area seemed to be key for the AuNPs, with the best TOF values obtained for the bioconjugates with AuNPs in the lid site. Finally, the protein environment and the asymmetric properties of the AuNPs were tested in the reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. A high reductive conversion and an enantiomeric excess of up to 39% towards (R)-1-phenylethanol was found using Au-Mt@GTL-114 pH 10 as a catalyst. Moderate enantioselectivity towards the opposite isomer was also observed using the Au-Mt@GTL-193 pH 10 conjugate.

De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery.

Conserved cysteine frameworks are essential components of disulfide-rich peptides (DRPs), which dominantly define the structural diversity of both naturally occurring and de novo-designed DRPs. However, there are only very limited numbers of conserved cysteine frameworks, and general methods enabling de novo discovery of cysteine frameworks with robust foldability are still not available. Here, we devised a "touchstone"-based strategy that relies on chasing oxidative foldability between two individual disulfide-rich folds on the phage surface to discover new cysteine frameworks from random sequences. Unique cysteine frameworks with a high degree of compatibility with phage display systems and broad sequence tolerance were successfully identified, which were subsequently exploited for the development of multicyclic DRP libraries, enabling the rapid discovery of new peptide ligands with low-nanomolar and picomolar binding affinity. This study provides an unprecedented method for exploring and exploiting the sequence and structure space of DRPs that is not readily accessible by existing strategies, holding the potential to revolutionize the study of DRPs and significantly advance the design and discovery of multicyclic peptide ligands and drugs.

50 shades of oxidative stress: A state-specific cysteine redox pattern hypothesis

Oxidative stress is biochemically complex. Like primary colours, specific reactive oxygen species (ROS) and antioxidant inputs can be mixed to create unique “shades” of oxidative stress. Even a minimal redox module comprised of just 12 (ROS & antioxidant) inputs and 3 outputs (oxidative damage, cysteine-dependent redox-regulation, or both) yields over half a million “shades” of oxidative stress. The present paper proposes the novel hypothesis that: state-specific shades of oxidative stress, such as a discrete disease, are associated with distinct tell-tale cysteine oxidation patterns. The patterns are encoded by many parameters, from the identity of the oxidised proteins, the cysteine oxidation type, and magnitude. The hypothesis is conceptually grounded in distinct ROS and antioxidant inputs coalescing to produce unique cysteine oxidation outputs. And considers the potential biological significance of the holistic cysteine oxidation outputs. The literature supports the existence of state-specific cysteine oxidation patterns. Measuring and manipulating these patterns offer promising avenues for advancing oxidative stress research. The pattern inspired hypothesis provides a framework for understanding the complex biochemical nature of state-specific oxidative stress.

Gonadotropic activity of a second relaxin-type peptide in starfish

In starfish, a relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropin that triggers gamete maturation and spawning. In common with other relaxin/insulin superfamily peptides, RGP consists of an A- and a B-chain, with cross-linkages mediated by one intra- and two inter-chain disulfide bonds. In this study, a second relaxin-like peptide (RLP2) was identified in starfish species belonging to the orders Valvatida, Paxillosida, and Forcipulatida. Like RGP, RLP2 precursors comprise a signal peptide and a C-peptide in addition to the A- and B-chains. However, a unique cysteine motif [CC-(3X)-C-(10X)-C] is present in the A-chain of RLP2, which contrasts with the cysteine motif in other members of the relaxin/insulin superfamily [CC-(3X)-C-(8X)-C]. Importantly, in vitro pharmacological tests revealed that Patiria pectinifera RLP2 (Ppe-RLP2) and Asterias rubens RLP2 (Aru-RLP2) trigger shedding of mature eggs from ovaries of P. pectinifera and A. rubens, respectively. Furthermore, the potencies of Ppe-RLP2 and Aru-RLP2 as gonadotropic peptides were similar to those of Ppe-RGP and Aru-RGP, respectively, and the effect of RLP2 exhibited partial species-specificity. These findings indicate that two relaxin-type peptides regulate spawning in starfish and therefore we propose that RGP and RLP2 are renamed RGP1 and RGP2, respectively.

Proteome-wide structural analysis identifies warhead- and coverage-specific biases in cysteine-focused chemoproteomics

Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on- and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.

Oxiforms: Unique cysteine residue- and chemotype-specified chemical combinations can produce functionally-distinct proteoforms: Like how mixing primary colours creates new shades, cysteine residue- and chemotype-specified chemical combinations can produce functionally-distinct proteoforms called oxiforms: Like how mixing primary colours creates new shades, cysteine residue- and chemotype-specified chemical combinations can produce functionally-distinct proteoforms called oxiforms.

A single protein molecule with one or more cysteine residues can occupy a plurality of unique residue and oxidation-chemotype specified proteoforms that I term oxiforms. In binary reduced or oxidised terms, one molecule with three cysteines will adopt one of eight unique oxiforms. Residue-defined sulfur chemistry endows specific oxiforms with distinct functionally-relevant biophysical properties (e.g., steric effects). Their emergent complexity means a functionally-relevant effect may only manifest when multiple cysteines are oxidised. Like how mixing colours makes new shades, combining discrete redox chemistries-colours-can create a kaleidoscope of oxiform hues. The sheer diversity of oxiforms co-existing within the human body provides a biological basis for redox heterogeneity. Of evolutionary significance, oxiforms may enable individual cells to mount a broad spectrum of responses to the same stimulus. Their biological significance, however plausible, is speculative because protein-specific oxiforms remain essentially unexplored. Excitingly, pioneering new techniques can push the field into uncharted territory by quantifying oxiforms. The oxiform concept can advance our understanding of redox-regulation in health and disease.

Anti-HER2 cell specific doxorubicin delivery using synthetic bacterial spores for resistant ovarian cancer treatment.

Abstract Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. SSHELs are synthetic spore-like particles wherein the spore’s cell surface is partially reconstituted around 1 μm-diameter silica beads. Via a unique cysteine engineered protein, the surface of SSHELs may be covalently decorated to display HER2 (SSHELs αHER2). SKOV3 and SKBR3 cells’ targeting by SSHELs αHER2was evaluated by flow cytometry and confocal microscopy. SSHELs αHER2specifically bound to target cells and were internalized proportionally to their concentration. Subsequently, SSHELs were successfully loaded with doxorubicin (Dox-SSHELs αHER2). Dox-SSHELs αHER2capabilities to kill tumor cells was evaluated by flow cytometry and caspase assay. HER2 negative cells were used as a control. Dox-SSHELs αHER2’s efficacy was tested in vivo using athymic nude mice injected with SKOV3 ovarian cancer cells and then treated for up to 40 days. Dox-SSHELs αHER2can reduce SKOV3 tumor growth up to 75% when compared to the free drug. Further, when compared to liposomal doxorubicin (Doxil TM) Dox-SSHELs αHER2showed similar efficacy in reducing tumor growth, but without any of the typical side effects correlated with Doxil TMinjections. With a facile, reproducible manufacturing process that allows for straightforward targeting towards specific cell types, and lower overall toxicity, we suggest that SSHELs may represent a versatile strategy opening new research avenues for targeted drug delivery treatment or immune system stimulation through vaccination.

Bifidobacterial GH146 β-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-β-l-Araf-(1→2)-β-l-Araf-(1→2)-β-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 β-l-arabinobiosidase (HypBA2), producing β-l-Araf-(1→2)-l-Ara (β-l-arabinobiose) and mono-β-l-Araf linked to the HRGP backbone. In bifidobacteria, the β-l-arabinobiose is then hydrolyzed by GH127 β-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 β-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

Designed ankyrin repeat proteins for detecting prostate-specific antigen expression in vivo.

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with a hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 ± 0.58% ID g-1 (n = 3; 2 h post-administration) and was reduced by ∼50% by competitive binding with a low molar activity formulation (blocking group: 2.47 ± 0.42% ID g-1; n = 3; P value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

In silico study of selective inhibition mechanism of S-adenosyl-L-methionine analogs for human DNA methyltransferase 3A

Epigenetic mechanisms leading to transcriptional regulation, including DNA methylation, are frequently dysregulated in diverse cancers. Interfering with aberrant DNA methylation performed by DNA cytosine methyltransferases (DNMTs) is a clinically validated approach. In particular, the selective inhibition of the de novo DNMT3A and DNMT3B enzymes, whose expression is limited to early embryogenesis, adult stem cells, and in cancers, is particularly attractive; such selectivity is likely to attenuate the dose limiting toxicity shown by current, non-selective DNMT inhibitors. We use molecular dynamics (MD) based computational analysis to study known small molecule binders of DNMT3A, then propose reversible, tight binding, and selective inhibitors that exploit the Asn1192/Arg688 difference between the maintenance DNMT1 and DNMT3A near the active site. A similar strategy exploiting the presence of a unique active site cysteine Cys666 is used to propose DNMT3A-selective irreversible inhibitors. We report our results of relative binding energies of the known and proposed compounds estimated using MM/GBSA and umbrella sampling (US) techniques, and our evaluation of other end-point binding free energy calculation methods for these receptors. These calculations offer insight into the potential for small molecules to selectively target the active site of DNMT3A.