Cysteine Residues Research Articles

The Power of Molecular Dynamics Simulations and Their Applications to Discover Cysteine Protease Inhibitors.

A large family of enzymes with the function of hydrolyzing peptide bonds, called peptidases or cysteine proteases (CPs), are divided into three categories according to the peptide chain involved. CPs catalyze the hydrolysis of amide, ester, thiol ester, and thioester peptide bonds. They can be divided into several groups, such as papain-like (CA), viral chymotrypsin-like CPs (CB), papain-like endopeptidases of RNA viruses (CC), legumain-type caspases (CD), and showing active residues of His, Glu/Asp, Gln, Cys (CE). The catalytic mechanism of CPs is the essential cysteine residue present in the active site. These mechanisms are often studied through computational methods that provide new information about the catalytic mechanism and identify inhibitors. The role of computational methods during drug design and development stages is increasing. Methods in Computer-Aided Drug Design (CADD) accelerate the discovery process, increase the chances of selecting more promising molecules for experimental studies, and can identify critical mechanisms involved in the pathophysiology and molecular pathways of action. Molecular dynamics (MD) simulations are essential in any drug discovery program due to their high capacity for simulating a physiological environment capable of unveiling significant inhibition mechanisms of new compounds against target proteins, especially CPs. Here, a brief approach will be shown on MD simulations and how the studies were applied to identify inhibitors or critical information against cysteine protease from several microorganisms, such as Trypanosoma cruzi (cruzain), Trypanosoma brucei (rhodesain), Plasmodium spp. (falcipain), and SARS-CoV-2 (Mpro). We hope the readers will gain new insights and use our study as a guide for potential compound identifications using MD simulations.

Identification of odorant binding protein and chemosensory protein genes in Protegira songi (Lepidoptera Noctuidae) via transcriptome analysis

The larvae of Protegira songi Chen & Zhang are notorious defoliator specifically infesting the medicinal plant Eucommia ulmoides Oliver. In insects, Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) are highly expressed in olfactory organs and play essential roles in perceiving female pheromone and host volatiles by binding and transporting odorant compounds. In this study, 17 OBPs and 16 CSPs were identified from P. songi by transcriptome analysis. Sequence alignment indicated that PsonOBPs and PsonCSPs share no identity with each other in protein sequence. The overall structures of PsonOBPs and PsonCSPs are mainly constructed by α-helix bundles and loops. But the distribution of conserved cysteine residues and arrangement of α-helix are varied between PsonOBPs and PsonCSPs. Putative ligand binding cavities were found in these predicted protein structures. Expression profiles of PsonOBPs and PsonCSPs in adults and larvae were determined by RT-PCR which showed that 16 PsonOBPs and 11 PsonCSPs were expressed in adult antennae or/and larval head. PsonGOBPs, PsonPBPs, PsonOBP5, PsonOBP7, and PsonCSP6 were preferentially expressed in olfactory associated tissues. Our results suggest that the PsonOBPs and PsonCSPs that contain ligand cavity and are highly enriched in olfactory tissues may play roles in chemosensory perception of P. songi, and the transcriptome data may facilitate the studies on the olfactory mechanism of P. songi specifically choosing E. ulmoides as host.

Deeper Insights into Mixed Crowding through Enzyme Activity, Dynamics, and Crowder Diffusion.

Given the fact that the cellular interior is crowded by many different kinds of macromolecules, it is important that in vitro studies be carried out in the presence of mixed crowder systems. In this regard, we have used binary crowders formed by the combination of some of the commonly used crowding agents, namely, Ficoll 70, Dextran 70, Dextran 40, and PEG 8000 (PEG 8), to study how these affect enzyme activity, dynamics, and crowder diffusion. The enzyme chosen is AK3L1, an isoform of adenylate kinase. To investigate its dynamics, we have carried out three single point mutations (A74C, A132C, and A209C) with the cysteine residues being labeled with a coumarin-based solvatochromic probe [CPM: (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin)]. Both enzyme activity and dynamics decreased in the binary mixtures as compared with the sum of the individual crowders, suggesting a reduction in excluded volume (in the mixture). To gain deeper insights into the binary mixtures, fluorescence correlation spectroscopy studies were carried out using fluorescein isothiocyanate-labeled Dextran 70 and tetramethylrhodamine-labeled AK3L1 as the diffusion probes. Diffusion in binary mixtures was observed to be much more constrained (relative to the sum of the individual crowders) for the labeled enzyme as compared to the labeled crowder showing different environments being faced by the two species. This was further confirmed during imaging of the phase-separated droplets formed in the binary mixtures having PEG as one of the crowding agents. The interior of these droplets was found to be rich in crowders and densely packed, as shown by confocal and digital holographic microscopy images, with the enzymes predominantly residing outside these droplets, that is, in the relatively less crowded regions. Taken together, our data provide important insights into various aspects of the simplest form of mixed crowding, that is, composed of just two components, and also hint at the enhanced complexity that the cellular interior presents toward having a detailed and comprehensive understanding of the same.

Comparison of Cyclic and Linear PEG Conjugates.

Bioconjugation of polymers to proteins is a method to impart improved stability and pharmacokinetic properties to biologic systems. However, the precise effects of polymer architecture on the resulting bioconjugates are not well understood. Particularly, cyclic polymers are known to possess unique features such as a decreased hydrodynamic radius when compared to their linear counterparts of the same molecular weight, but have not yet been studied. Here, we report the first bioconjugation of a cyclic polymer, poly(ethylene glycol) (PEG), to a model protein, T4 lysozyme, containing a single engineered cysteine residue (V131C). We compare the stability and activity of this conjugate with those of a linear PEG-T4 lysozyme analogue of similar molecular weight. Furthermore, we used molecular dynamics (MD) simulations to determine the behavior of the polymer-protein conjugates in solution. We introduce cyclic polymer-protein conjugates as potential candidates for the improvement of biologic therapeutics.

Is autism a PIN1 deficiency syndrome? A proposed etiological role for glyphosate.

Autism is a neurodevelopmental disorder, the prevalence of which has increased dramatically in the United States over the past two decades. It is characterized by stereotyped behaviors and impairments in social interaction and communication. In this paper, we present evidence that autism can be viewed as a PIN1 deficiency syndrome. Peptidyl-prolyl cis/trans isomerase, NIMA-Interacting 1 (PIN1) is a peptidyl-prolyl cis/trans isomerase, and it has widespread influences in biological organisms. Broadly speaking, PIN1 deficiency is linked to many neurodegenerative diseases, whereas PIN1 over-expression is linked to cancer. Death-associated protein kinase 1 (DAPK1) strongly inhibits PIN1, and the hormone melatonin inhibits DAPK1. Melatonin deficiency is strongly linked to autism. It has recently been shown that glyphosate exposure to rats inhibits melatonin synthesis as a result of increased glutamate release from glial cells and increased expression of metabotropic glutamate receptors. Glyphosate's inhibition of melatonin leads to a reduction in PIN1 availability in neurons. In this paper, we show that PIN1 deficiency can explain many of the unique morphological features of autism, including increased dendritic spine density, missing or thin corpus callosum, and reduced bone density. We show how PIN1 deficiency disrupts the functioning of powerful high-level signaling molecules, such as nuclear factor erythroid 2-related factor 2 (NRF2) and p53. Dysregulation of both of these proteins has been linked to autism. Severe depletion of glutathione in the brain resulting from chronic exposure to oxidative stressors and extracellular glutamate leads to oxidation of the cysteine residue in PIN1, inactivating the protein and further contributing to PIN1 deficiency. Impaired autophagy leads to increased sensitivity of neurons to ferroptosis. It is imperative that further research be conducted to experimentally validate whether the mechanisms described here take place in response to chronic glyphosate exposure and whether this ultimately leads to autism.

Evaluation of the Biological Activities of Peptides from Epidermal Mucus of Marine Fish Species from Chilean Aquaculture

The skin of fish is a physicochemical barrier that is characterized by being formed by cells that secrete molecules responsible for the first defense against pathogenic organisms. In this study, the biological activity of peptides from mucus of Seriola lalandi and Seriolella violacea were identified and characterized. To this purpose, peptide extraction was carried out from epidermal mucus samples of juveniles of both species, using chromatographic strategies for purification. Then, the peptide extracts were characterized to obtain the amino acid sequence by mass spectrometry. Using bioinformatics tools for predicting antimicrobial and antioxidant activity, 12 peptides were selected that were chemically produced by simultaneous synthesis using the Fmoc-Tbu strategy. The results revealed that the synthetic peptides presented a random coil or extended secondary structure. The analysis of antimicrobial activity allowed it to be discriminated that four peptides, named by their synthesis code 5065, 5069, 5070, and 5076, had the ability to inhibit the growth of Vibrio anguillarum and affected the copepodite stage of C. rogercresseyi. On the other hand, peptides 5066, 5067, 5070, and 5077 had the highest antioxidant capacity. Finally, peptides 5067, 5069, 5070, and 5076 were the most effective for inducing respiratory burst in fish leukocytes. The analysis of association between composition and biological function revealed that the antimicrobial activity depended on the presence of basic and aromatic amino acids, while the presence of cysteine residues increased the antioxidant activity of the peptides. Additionally, it was observed that those peptides that presented the highest antimicrobial capacity were those that also stimulated respiratory burst in leukocytes. This is the first work that demonstrates the presence of functional peptides in the epidermal mucus of Chilean marine fish, which provide different biological properties when the fish face opportunistic pathogens.

Anti-Inflammatory Therapeutic Mechanisms of Isothiocyanates: Insights from Sulforaphane

Isothiocyanates (ITCs) belong to a group of natural products that possess a highly reactive electrophilic -N=C=S functional group. They are stored in plants as precursor molecules, glucosinolates, which are processed by the tyrosinase enzyme upon plant tissue damage to release ITCs, along with other products. Isolated from broccoli, sulforaphane is by far the most studied antioxidant ITC, acting primarily through the induction of a transcription factor, the nuclear factor erythroid 2–related factor 2 (Nrf2), which upregulates downstream antioxidant genes/proteins. Paradoxically, sulforaphane, as a pro-oxidant compound, can also increase the levels of reactive oxygen species, a mechanism which is attributed to its anticancer effect. Beyond highlighting the common pro-oxidant and antioxidant effects of sulforaphane, the present paper was designed to assess the diverse anti-inflammatory mechanisms reported to date using a variety of in vitro and in vivo experimental models. Sulforaphane downregulates the expression of pro-inflammatory cytokines, chemokines, adhesion molecules, cycloxyhenase-2, and inducible nitric oxide synthase. The signalling pathways of nuclear factor κB, activator protein 1, sirtuins 1, silent information regulator sirtuin 1 and 3, and microRNAs are among those affected by sulforaphane. These anti-inflammatory actions are sometimes due to direct action via interaction with the sulfhydryl structural moiety of cysteine residues in enzymes/proteins. The following are among the topics discussed in this paper: paradoxical signalling pathways such as the immunosuppressant or immunostimulant mechanisms; crosstalk between the oxidative and inflammatory pathways; and effects dependent on health and disease states.

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors.

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

Chemical Synthesis of Secretoglobin 3A2 Covalent Homodimer and Photocaged Monomeric Variants

AbstractSecretoglobin (SCGB) 3A2 belongs to an intriguing family of small, secreted proteins present only in mammals. Although members of the SCGB protein family have distinct amino acid sequences, they share structural similarities. Of particularly interest is the not yet fully understood self‐assembly ability of SCGBs, which arise from covalent disulfide dimerization and non‐covalent oligomerization. Recently, SCGB3A2 has attracted attention for its singular expression profile in airways. However, the knowledge on SCGB3A2 (patho)physiology derives exclusively from in vivo and complex ex vivo mixtures, which hampers characterization of the mechanisms driving SCGB3A2 structural behavior. Herein, we document the chemical synthesis of SCGB3A2 in multi‐milligram quantities. Key to access both monomeric and homodimeric SCGB3A2 analogues was the use of KAHA ligation and enabled masking of the cysteine residue. The synthetic proteins were used to investigate the SCGB3A2 self‐assembly profile, confirming their high propensity to dimerization even in the absence of the key Cys residue.

A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

Molecular cloning, expression, purification and functional characterization of Rv1588c of Mycobacteriumtuberculosis

Biotin is an essential cofactor for biotin dependent enzymes necessary for synthesis of fatty acids including mycolic acids, an essential component of cell wall of Mycobacterium tuberculosis. Biotin is synthesized by the genes located in bio operons which are fairly conserved in mycobacteria. Biotin synthesis is controlled intracellularly in Mycobacterium smegmatis by the gene bioQ, located opposite to bioB. Such locations of biotin regulators are found to be conserved among various mycobacteria species. In M. tuberculosis, rv1588c is such an uncharacterized gene, located opposite to bioB which shares limited similarity with DNA-binding region of BioQ of M. smegmatis, a TetR-type protein. A highly similar M. smegmatis-like BioQ binding site is also present within the bioB promoter of M. tuberculosis. This suggests that Rv1588c might be the BioQ for M. tuberculosis. The present study reports in-silico and functional characterization of Rv1588c. The structure of Rv1588c has been partially modelled and validated computationally. The N-terminal domain of Rv1588c is found to be structurally similar with DNA-binding domains of several transcriptional regulators. rv1588c gene has been cloned, expressed, and purified. Limited proteolysis study revealed presence of C-terminal domain in Rv1588c which indicates presence of a two-domain structure, like those of canonical transcriptional regulators. Rv1588c forms significant amount of dimers in solution, mediated by cysteine residues. Purified Rv1588c binds with bioB promoter region in-vitro. This suggests that Rv1588c is probably the transcriptional regulator that might control the expression of bioB of M. tuberculosis.

Binding Mechanism between Platelet Glycoprotein and Cyclic Peptide Elucidated by McMD-Based Dynamic Docking.

The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the platelet glycoprotein Ibα (GPIbα) and the von Willebrand factor (vWF) by forming a complex with GPIbα. To study the binding mechanism between GPIbα and OS1 and, therefore, the inhibition mechanism of the protein-protein GPIbα-vWF complex, we have applied our multicanonical molecular dynamics (McMD)-based dynamic docking protocol starting from the unbound state of the peptide. Our simulations have reproduced the experimental complex structure, although the top-ranking structure was an intermediary one, where the peptide was bound in the same location as in the experimental structure; however, the β-switch of GPIbα attained a different conformation. Our analysis showed that subsequent refolding of the β-switch results in a more stable binding configuration, although the transition to the native configuration appears to take some time, during which OS1 could dissociate. Our results show that conformational changes in the β-switch are crucial for successful binding of OS1. Furthermore, we identified several allosteric binding sites of GPIbα that might also interfere with vWF binding, and optimization of the peptide to target these allosteric sites might lead to a more effective inhibitor, as these are not dependent on the β-switch conformation.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKβ independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Finding the Ajoene Sweet-Spot: Structure-Activity Relations that Govern its Blood Stability and Cancer Cytotoxicity.

Ajoene is an organosulfur compound found in crushed garlic that exerts its anti-cancer activity by S-thiolating cysteine residues on proteins. Its development is hampered due to limited bioavailability, so in this study, we synthesised analogues of ajoene to probe the significance of the ajoene vinyl disulfide/sulfoxide core with respect to cytotoxicity and blood stability. Polar side groups were also incorporated to improve aqueous solubility. It was found that derivatives containing a vinyl disulfide functional group (4-7, as in ajoene), were more cytotoxic compared to analogues in which the double bond was removed, although the latter showed superior blood stability. It was also found that the allyl-S sulfur of the disulfide was more electrophilic to S-thiolysis based on the global electrophilicity index (ω) and the condensed electrophilic Fukui function . S-Thiolysis was found to be exergonic for the vinyl disulfides based on entropy and enthalpy computations with a deprotonated thiolate. Derivatisation to the dihydro (10, 12) and deoxydihydroajoenes (9, 11) produced analogues that were slightly less potent but with greatly improved blood stability. Taken together, the deoxydihydroajoenes present themselves as good candidates for further therapeutic development.

Inducing α-Helicity in Peptides by Silver Coordination to Cysteine.

Short peptide sequences consisting of two cysteine residues separated by three other amino acids display complete change from random coil to α-helical secondary structure in response to addition of Ag+ ions. The folded CXXXC/Ag+ complex involves formation of multinuclear Ag+ species and is stable in a wide pH range from below 3 to above 8. The complex is stable through reversed-phase HPLC separation as well as towards a physiological level of chloride ions, based on far-UV circular dichroism spectroscopy. In electrospray MS under acidic conditions a peptide dimer with four Ag+ ions bound was observed, and modelling based on potentiometric experiments supported this to be the dominating complex at neutral pH together with a peptide dimer with 3 Ag+ and one proton at lower pH. The complex was demonstrated to work as a N-terminal nucleation site for inducing α-helicity into longer peptides. This type of silver-mediated peptide assembly and folding may be of more general use for stabilizing not only peptide folding but also for controlling oligomerization even under acidic conditions.

Immunoglobulin Superfamily Containing Leucine-Rich Repeat (ISLR) Serves as a Redox Sensor That Modulates Antioxidant Capacity by Suppressing Pyruvate Kinase Isozyme M2 Activity.

Cells defend against oxidative stress by enhancing antioxidant capacity, including stress-activated metabolic alterations, but the underlying intracellular signaling mechanisms remain unclear. This paper reports that immunoglobulin superfamily containing leucine-rich repeat (ISLR) functions as a redox sensor that responds to reactive oxygen species (ROS) stimulation and modulates the antioxidant capacity by suppressing pyruvate kinase isozyme M2 (PKM2) activity. Following oxidative stress, ISLR perceives ROS stimulation through its cysteine residue 19, and rapidly degrades in the autophagy-lysosome pathway. The downregulated ISLR enhances the antioxidant capacity by promoting the tetramerization of PKM2, and then enhancing the pyruvate kinase activity, PKM2-mediated glycolysis is crucial to the ISLR-mediated antioxidant capacity. In addition, our results demonstrated that, in triple-negative breast cancer, cisplatin treatment reduced the level of ISLR, and PKM2 inhibition sensitizes tumors to cisplatin by enhancing ROS production; and argued that PKM2 inhibition can synergize with cisplatin to limit tumor growth. Our results demonstrate a molecular mechanism by which cells respond to oxidative stress and modulate the redox balance.

Synthetic ShK-like Peptide from the Jellyfish Nemopilema nomurai Has Human Voltage-Gated Potassium-Channel-Blocking Activity.

We identified a new human voltage-gated potassium channel blocker, NnK-1, in the jellyfish Nemopilema nomurai based on its genomic information. The gene sequence encoding NnK-1 contains 5408 base pairs, with five introns and six exons. The coding sequence of the NnK-1 precursor is 894 nucleotides long and encodes 297 amino acids containing five presumptive ShK-like peptides. An electrophysiological assay demonstrated that the fifth peptide, NnK-1, which was chemically synthesized, is an effective blocker of hKv1.3, hKv1.4, and hKv1.5. Multiple-sequence alignment with cnidarian Shk-like peptides, which have Kv1.3-blocking activity, revealed that three residues (3Asp, 25Lys, and 34Thr) of NnK-1, together with six cysteine residues, were conserved. Therefore, we hypothesized that these three residues are crucial for the binding of the toxin to voltage-gated potassium channels. This notion was confirmed by an electrophysiological assay with a synthetic peptide (NnK-1 mu) where these three peptides were substituted with 3Glu, 25Arg, and 34Met. In conclusion, we successfully identified and characterized a new voltage-gated potassium channel blocker in jellyfish that interacts with three different voltage-gated potassium channels. A peptide that interacts with multiple voltage-gated potassium channels has many therapeutic applications in various physiological and pathophysiological contexts.

Enhanced EphB2-Specific Peptide Inhibitors through Stabilization of Polyproline II Helical Structure.

Ephrin (Eph) receptors are the largest family of receptor tyrosine kinases. Interactions between Eph receptors and their membrane-bound ephrin protein ligands are associated with many developmental processes as well as various cancers and neurodegenerative diseases. With significant crosstalk between different Eph receptors and ephrin ligands, there is an urgent need for high-affinity ligands that bind specifically to individual Eph receptors to interrogate and modulate their functions. Here, we describe the rational development of potent EphB2 receptor inhibitors derived from the EphB2 receptor-specific SNEW peptide. To improve inhibitory potency, we evaluated 20+ cross-linkers with the goal of spanning and stabilizing a single polyproline II helical turn observed when SNEW binds to the EphB2 receptor. Of the cross-linkers evaluated, an 11-atom cross-linker, composed of a rigid 2,7-dimethylnaphthyl moiety between two cysteine residues, was found to yield the most potent inhibitor. Analysis of the cyclized region of this peptide by NMR and molecular dynamics simulations suggests that cross-linking stabilizes the receptor-bound polyproline II helix structure observed in the receptor-peptide complex. Cross-linked SNEW variants retained binding specificity for EphB2 and showed cross-linker-dependent resistance to trypsin proteolysis. Beyond the discovery of more potent EphB2 receptor inhibitors, these studies illustrate a novel cyclization approach with potential to stabilize polyproline II helical structure in various peptides for specific targeting of the myriad protein-protein interactions (PPIs) mediated by polyproline II helices.

Formation of the Calpain-1/Calpastatin Complex Promotes Activation of Calpain-1 under Oxidizing Conditions.

Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (N-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165mM or 295mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG;MW=5000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decrased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.

Targeting KRAS Oncogene for Patients With Colorectal Cancer: A New Step Toward Precision Medicine.

KRAS mutations are common driver oncogenes associated with the development of several solid tumors. KRAS oncogene has been considered a highly challenging target for drug development because of structural features, including the lack of deep groove on its catalytic unit. However, by leveraging cysteine residues, covalent KRAS inhibitors irreversibly trap KRAS G12C mutants in their inactive GDP-bound state. These agents have resulted in significant clinical responses among patients with KRAS G12C-mutant solid tumors, including patients with colorectal cancer (CRC). Other allele-specific inhibitors of KRAS oncogene and panKRAS and panRAS inhibitors are also currently being investigated in clinical trials. This review article overviews recent clinical progress on KRAS G12C targeting for the management of patients with KRAS G12C-mutant CRC and provides an update on other RAS targeting approaches. We also discuss the unique biological features of RAS-mutant CRC, which require the combination of KRAS inhibitors and anti-epidermal growth factor receptor therapy, and elaborate on resistance mechanisms and novel therapeutic avenues that may define future treatment paradigms of patients with RAS-mutant CRC.