A mouse model of sensory neuropathy induced by a long course of monomethyl-auristatin E treatment.

Antibody-drug conjugates (ADCs) are anticancer drugs consisting of a monoclonal antibody, targeting selective tumor antigens, to which has been frequently associated a highly potent cytotoxic agent, the monomethyl auristatin E (MMAE) using a chemical linker. MMAE is a tubulin polymerization inhibitor derived from dolastin-10. These MMAE-ADCs are responsible for peripheral nerve toxicities. Our objective was to develop and characterize a mouse model of MMAE-induced peripheral neuropathy induced by free MMAE injections. MMAE was injected in Swiss mice at 50 µg/kg i.p. every other day for 7 weeks. Assessments of motor and sensory nerve functions were performed once a week on MMAE and Vehicle-treated mice. Sciatic nerve and paw skin were removed at the end of experiment for subsequent immunofluorescence and morphological analysis. MMAE did not affect motor coordination, muscular strength and heat nociception, but significantly induced tactile allodynia in MMAE-treated mice compared with Vehicle-treated mice from day 35 to day 49. MMAE significantly reduced myelinated and unmyelinated axon densities in sciatic nerves and led to a loss of intraepidermal nerve fiber in paw skin. In summary, long course of low dose of MMAE induced a peripheral sensory neuropathy associated with nerve degeneration, without general state alteration. This model may represent a ready accessible tool to screen neuroprotective strategies in the context of peripheral neuropathies induced by MMAE-ADCs.

Synergistic toxicity of some cyanobacterial oligopeptides to physiological activities of Daphnia magna (Crustacea).

Anabaenopeptins and microcystins are oligopeptides produced by bloom-forming cyanobacteria. We determined in vivo effects of anabaenopeptin-B (AN-B) and two variants of microcystins of different hydrophobicity (MC-LR and MC-LF) on the physiology of Daphnia magna. Heart rate, thoracic limb activity and post-abdominal claw activity were determined by digital video analysis and oxygen consumption by Oxygraph + system. EC50 calculation and isobole methodology for interactive effects of AN-B and MC-LR mixture were used. Daphnids' responses to all three oligopeptides were concentration- and time-dependent. MC-LF was the most potent inhibitor of heart rate, thoracic limb activity, post-abdominal claw activity and oxygen consumption. AN-B was more toxic than MC-LR toward oxygen consumption; it inhibited the movements of limbs and post-abdominal claw similarly to MC-LR, but did not inhibit heart rate. The strongest toxic effects were induced by the binary mixture of AN-B with MC-LR at the sum concentration equal to the concentration of the single compounds. First time direct synergistic toxic effects of the cyanopeptides on all the physiological parameters were found. The obtained results explain stronger disturbances in aquatic organisms caused by cyanobacterial cell contents than the individual cyanopeptides present even at higher concentrations. Other metabolites and their interactions need further studies.

Gold Nanorod Enhanced Photoacoustic Microscopy and Optical Coherence Tomography of Choroidal Neovascularization.

Visualization and evaluation of choroidal neovascularization (CNV) are major challenges to improve treatment outcomes for patients with age-related macular degeneration (AMD). Limitations of current imaging techniques include the limited penetration depth, spatial resolution, and sensitivity and difficulty visualizing CNV from the healthy microvasculature. In this study, a custom-built multimodal photoacoustic microscopy (PAM) and optical coherence tomography (OCT) system was developed to distinguish the margin of CNV in living rabbits with the assistance of functionalized gold nanorods conjugating with RGD ligands (GNR-RGD). Intravenous administration of GNR-RGD into rabbits in a CNV model resulted in signal enhancements of 27.2-fold in PAM and 171.4% in OCT. This molecular imaging technique of contrast-enhanced PAM and OCT is a promising tool for the precise imaging of CNV as well as the evaluation of the pathophysiology in vivo without destruction of tissue.

Modified histidine containing amphipathic ultrashort antifungal peptide, His[2-p-(n-butyl)phenyl]-Trp-Arg-OMe exhibits potent anticryptococcal activity.

In pursuit of ultrashort peptide-based antifungals, a new structural class, His(2-aryl)-Trp-Arg is reported. Structural changes were investigated on His-Trp-Arg scaffold to demonstrate the impact of charge and lipophilic character on the biological activity. The presence and size of the aryl moiety on imidazole of histidine modulated overall amphiphilic character, and biological activity. Peptides exhibited IC50 of 0.37-9.66 µg/mL against C. neoformans. Peptide 14f [His(2-p-(n-butyl)phenyl)-Trp-Arg-OMe] exhibited two-fold potency (IC50 = 0.37 µg/mL, MIC = 0.63 µg/mL) related to amphotericin B, without any cytotoxic effects up to 10 µg/mL. Peptide 14f act by nuclear fragmentation, membranes permeabilization, disruption and pore formations in the microbial cells as determined by the mechanistic studies employing Trp-quenching, CLSM, SEM, and HR-TEM. The amalgamation of short sequence, presence of appropriate aryl group on l-histidine, potent anticryptococcal activity, no cytotoxicity, and detailed mechanistic studies directed to the identification of 14f as a new antifungal structural lead.

Self-assembly of bio-inspired heterochiral peptides.

Peptide hydrogels, deriving from natural protein fragments, present unique advantages as compatibility and low cost of production that allow their wide application in different fields as wound healing, cell delivery and tissue regeneration. To engineer new biomaterials, the change of the chirality of single amino acids demonstrated a powerful approach to modulate the self-assembly mechanism. Recently we unveiled that a small stretch spanning residues 268-273 in the C-terminal domain (CTD) of Nucleophosmin 1 (NPM1) is an amyloid sequence. Herein, we performed a systematic D-scan of this sequence and analyzed the structural properties of obtained peptides. The conformational and kinetic features of self-aggregates and the morphologies of derived microstructures were investigated by means of different biophysical techniques, as well as the compatibility of hydrogels was evaluated in HeLa cells. All the investigated hexapeptides formed hydrogels even if they exhibited different conformational intermediates during aggregation, and they structural featured are finely tuned by introduced chiralities.

Tubulin binding potentially clears up Bortezomib and Carfilzomib differential neurotoxic effect.

Proteasome inhibitors (PIs) represent the gold standard in the treatment of multiple myeloma. Among PIs, Bortezomib (BTZ) is frequently used as first line therapy, but peripheral neuropathy (PN), occurring approximately in 50% of patients, impairs their life, representing a dose-limiting toxicity. Carfilzomib (CFZ), a second-generation PI, induces a significantly less severe PN. We investigated possible BTZ and CFZ off-targets able to explain their different neurotoxicity profiles. In order to identify the possible PIs off-targets we used the SPILLO-PBSS software that performs a structure-based in silico screening on a proteome-wide scale. Among the top-ranked off-targets of BTZ identified by SPILLO-PBSS we focused on tubulin which, by contrast, did not turn out to be an off-target of CFZ. We tested the hypothesis that the direct interaction between BTZ and microtubules would inhibit the tubulin alfa GTPase activity, thus reducing the microtubule catastrophe and consequently furthering the microtubules polymerization. This hypothesis was validated in a cell-free model, since BTZ (but not CFZ) reduces the concentration of the free phosphate released during GTP hydrolysis. Moreover, NMR binding studies clearly demonstrated that BTZ, unlike CFZ, is able to interact with both tubulin dimers and polymerized form. Our data suggest that different BTZ and CFZ neurotoxicity profiles are independent from their proteasome inhibition, as demonstrated in adult mice dorsal root ganglia primary sensory neurons, and, first, we demonstrate, in a cell free model, that BTZ is able to directly bind and perturb microtubules.

N-terminal selective conjugation method widens the therapeutic window of antibody-drug conjugates by improving tolerability and stability.

Antibody-drug conjugates (ADCs) are targeted therapeutic agents that treat cancers by selective delivery of highly potent cytotoxic drugs to tumor cells via cancer-specific antibodies. However, their clinical benefit is limited by off-target toxicity and narrow therapeutic windows. To overcome these limitations, we have applied reductive alkylation to develop a new type of ADC that has cytotoxic drugs conjugated to the N-terminal of an antibody through amine bonds introduced via reductive alkylation reactions (NTERM). To test whether the NTERM-conjugated ADCs can widen therapeutic windows, we synthesized three different ADCs by conjugating trastuzumab and monomethyl auristatin-F using three different methods, and compared their stability, efficacy, and toxicity. The NTERM-conjugated ADC was more stable in vitro and in vivo than the thiol-conjugated and the lysine-conjugated ADCs. The NTERM-conjugated ADC showed lower toxicity compared to other ADCs, whereas its efficacy was comparable to that of the thiol-conjugated ADC and better than that of the lysine-conjugated ADC. These results suggest that the NTERM conjugation method could widen the therapeutic window of ADCs by enhancing its stability and reducing toxicity.

Lipid/PAA-coated mesoporous silica nanoparticles for dual-pH-responsive codelivery of arsenic trioxide/paclitaxel against breast cancer cells.

Nanomedicine has attracted increasing attention and emerged as a safer and more effective modality in cancer treatment than conventional chemotherapy. In particular, the distinction of tumor microenvironment and normal tissues is often used in stimulus-responsive drug delivery systems for controlled release of therapeutic agents at target sites. In this study, we developed mesoporous silica nanoparticles (MSNs) coated with polyacrylic acid (PAA), and pH-sensitive lipid (PSL) for synergistic delivery and dual-pH-responsive sequential release of arsenic trioxide (ATO) and paclitaxel (PTX) (PL-PMSN-PTX/ATO). Tumor-targeting peptide F56 was used to modify MSNs, which conferred a target-specific delivery to cancer and endothelial cells under neoangiogenesis. PAA- and PSL-coated nanoparticles were characterized by TGA, TEM, FT-IR, and DLS. The drug-loaded nanoparticles displayed a dual-pH-responsive (pHe = 6.5, pHendo = 5.0) and sequential drug release profile. PTX within PSL was preferentially released at pH = 6.5, whereas ATO was mainly released at pH = 5.0. Drug-free carriers showed low cytotoxicity toward MCF-7 cells, but ATO and PTX co-delivered nanoparticles displayed a significant synergistic effect against MCF-7 cells, showing greater cell-cycle arrest in treated cells and more activation of apoptosis-related proteins than free drugs. Furthermore, the extracellular release of PTX caused an expansion of the interstitial space, allowing deeper penetration of the nanoparticles into the tumor mass through a tumor priming effect. As a result, FPL-PMSN-PTX/ATO exhibited improved in vivo circulation time, tumor-targeted delivery, and overall therapeutic efficacy.

Incidence and Management of Carfilzomib-induced Cardiovascular Toxicity; A Systematic Review and Meta-analysis.

BACKGROUND: The ASPIRE and ENDEAVOUR trials have shown cardiovascular adverse effects in patients treated with carfilzomib-based regimens. Therefore, we conducted this meta- analysis of published clinical trials to identify the cumulative incidence and risk of cardiovascular adverse effects due to carfilzomib. METHODS: A systematic search of PubMed, Embase, Web of Science, and Cochrane library was performed, and we identified 45 prospective trials of carfilzomib with data on 5583 patients. Among all patients being treated with carfilzomib (N=5,583), 8.9% sustained all grade cardiotoxicity, while 4.4% sustained high-grade cardiotoxicity. All-grade hypertension was present in 13.2%, while the incidence of high-grade hypertension was 5.3%. RESULTS: The observed incidences of all-grade heart failure, edema, and ischemia were 5.1%, 20.7%, and 4.6%, respectively. Likewise, for high-grade heart failure and edema observed incidence was 3.2%, and 2.7%, respectively. There was no difference in the event rate of all and highgrade cardiotoxicity between newly diagnosed multiple myeloma and relapsed/refractory (p-value 0.42 and 0.86, respectively). Likewise, we did not observe any difference in the event rate of all and high-grade cardiotoxicity when carfilzomib was used as a single agent versus when used in combination therapy with other agents (p-value 0.43 and 0.73, respectively). CONCLUSION: Carfilzomib is associated with a significant risk of cardiovascular toxicity and hypertension. With the increasing utilization of carfilzomib, it is critical for primary care physicians, oncologists and cardiologists to be aware of the risk of cardiotoxicity associated with the use of carfilzomib to recognize and treat baseline cardiovascular risk factors in such patients.

Microtubule and tubulin binding and regulation of microtubule dynamics by the antibody drug conjugate (ADC) payload, monomethyl auristatin E (MMAE): Mechanistic insights into MMAE ADC peripheral neuropathy.

Monomethyl auristatin E (MMAE) is a potent anti-cancer microtubule-targeting agent (MTA) used as a payload in three approved MMAE-containing antibody drug conjugates (ADCs) and multiple ADCs in clinical development to treat different types of cancers. Unfortunately, MMAE-ADCs can induce peripheral neuropathy, a frequent adverse event leading to treatment dose reduction or discontinuation and subsequent clinical termination of many MMAE-ADCs. MMAE-ADC-induced peripheral neuropathy is attributed to non-specific uptake of the ADC in peripheral nerves and release of MMAE, disrupting microtubules (MTs) and causing neurodegeneration. However, molecular mechanisms underlying MMAE and MMAE-ADC effects on MTs remain unclear. Here, we characterized MMAE-tubulin/MT interactions in reconstituted in vitro soluble tubulin or MT systems and evaluated MMAE and vcMMAE-ADCs in cultured human MCF7 cells. MMAE bound to soluble tubulin heterodimers with a maximum stoichiometry of ~1:1, bound abundantly along the length of pre-assembled MTs and with high affinity at MT ends, introduced structural defects, suppressed MT dynamics, and reduced the kinetics and extent of MT assembly while promoting tubulin ring formation. In cells, MMAE and MMAE-ADC (via nonspecific uptake) suppressed proliferation, mitosis and MT dynamics, and disrupted the MT network. Comparing MMAE action to other MTAs supports the hypothesis that peripheral neuropathy severity is determined by the precise mechanism(s) of each individual drug-MT interaction (location of binding, affinity, effects on morphology and dynamics). This work demonstrates that MMAE binds extensively to tubulin and MTs and causes severe MT dysregulation, providing convincing evidence that MMAE-mediated inhibition of MT-dependent axonal transport leads to severe peripheral neuropathy.

A prostate-specific membrane antigen (PSMA)-targeted prodrug with a favorable in vivo toxicity profile.

Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed a non-radioactive prodrug, SBPD-1, composed of a small-molecule PSMA-targeting moiety, a cancer-selective cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SBPD-1 demonstrated high binding affinity to PSMA (Ki = 8.84 nM) and selective cytotoxicity to PSMA-expressing PC cell lines (IC50 = 3.90 nM). SBPD-1 demonstrated a significant survival benefit in two murine models of human PC relative to controls. The highest dose tested did not induce toxicity in immunocompetent mice. The high specific targeting ability of SBPD-1 to PSMA-expressing tumors and its favorable toxicity profile warrant its further development.

Pilot Study: Quantitative Photoacoustic Evaluation of Peripheral Vascular Dynamics Induced by Carfilzomib In Vivo.

Carfilzomib is mainly used to treat multiple myeloma. Several side effects have been reported in patients treated with carfilzomib, especially those associated with cardiovascular events, such as hypertension, congestive heart failure, and coronary artery disease. However, the side effects, especially the manifestation of cardiovascular events through capillaries, have not been fully investigated. Here, we performed a pilot experiment to monitor peripheral vascular dynamics in a mouse ear under the effects of carfilzomib using a quantitative photoacoustic vascular evaluation method. Before and after injecting the carfilzomib, bortezomib, and PBS solutions, we acquired high-resolution three-dimensional PAM data of the peripheral vasculature of the mouse ear during each experiment for 10 h. Then, the PAM maximum amplitude projection (MAP) images and five quantitative vascular parameters, i.e., photoacoustic (PA) signal, diameter, density, length fraction, and fractal dimension, were estimated. Quantitative results showed that carfilzomib induces a strong effect on the peripheral vascular system through a significant increase in all vascular parameters up to 50%, especially during the first 30 min after injection. Meanwhile, bortezomib and PBS do not have much impact on the peripheral vascular system. This pilot study verified PAM as a comprehensive method to investigate peripheral vasculature, along with the effects of carfilzomib. Therefore, we expect that PAM may be useful to predict cardiovascular events caused by carfilzomib.

In Vivo Effect of RSH-12, a Novel Selective MMP-9 Inhibitor Peptide, in the Treatment of Experimentally Induced Dry Eye Model.

PURPOSE: To investigate the efficacy of RSH-12, a novel selective matrix metalloproteinase 9 (MMP-9) inhibitor peptide in rabbit models of dry eye syndrome (DES). METHODS: In vitro toxicity of RSH-12 on cultured human corneal fibroblasts was investigated with MTT. Ocular toxicity of RSH-12 was investigated by clinical examinations, histology, and TUNEL assay. Experimental model of dry eye was induced by 1.0% atropine sulfate administration followed after 15 min by treatment with PBS, RSH-12, and Restasis in individual groups, three times a day for 7 days. In addition to performing Schirmer's test for evaluating basic tear secretion and tear break-up time test for investigating tear stability, the occurrence of superficial punctate keratopathy was also investigated in the study groups. RESULTS: MTT assay demonstrated that RSH-12 was not toxic to human corneal fibroblasts in different concentrations. During the administration of atropine, TBUT values and tear volume were decreased in vehicle group while these indices improved significantly in groups treated with RSH-12 in a promising manner. RSH-12 increased the mean value of tear volume from 4.85 to 10.75 mm (P = .0001) and mean of TBUT values from 20.3 s to 34.5 s (P = .0001) compared with the vehicle. In contrast to the presence of severe superficial punctate keratopathy in the controls, no significant dotted staining was observed in the RSH-12 and Restasis groups. CONCLUSIONS: These outcomes propose that RSH-12 has a therapeutic effect in the rabbit model of dry eye and might be a potential treatment for severe DES.

Alternating Cationic-Hydrophobic Peptide/Peptoid Hybrids: Influence of Hydrophobicity on Antibacterial Activity and Cell Selectivity.

The influence of hydrophobicity on antibacterial activity versus the effect on the viability of mammalian cells for peptide/peptoid hybrids was examined for oligomers based on the cationic Lys-like peptoid residue combined with each of 28 hydrophobic amino acids in an alternating sequence. Their relative hydrophobicity was correlated to activity against both Gram-negative and Gram-positive species, human red blood cells, and HepG2 cells. This identified hydrophobic side chains that confer potent antibacterial activity (e. g., MICs of 2-8 µg/mL against E. coli) and low toxicity toward mammalian cells (<10 % hemolysis at 400 µg/mL and IC50 >800 µg/mL for HepG2 viability). Most peptidomimetics retained activity against drug-resistant strains. These findings corroborate the hypothesis that for related peptidomimetics two hydrophobicity thresholds may be identified: i) it should exceed a certain level in order to confer antibacterial activity, and ii) there is an upper limit, beyond which cell selectivity is lost. It is envisioned that once identified for a given subclass of peptide-like antibacterials such thresholds can guide further optimisation.

Investigating the Vascular Toxicity Outcomes of the Irreversible Proteasome Inhibitor Carfilzomib.

BACKGROUND: Carfilzomib's (Cfz) adverse events in myeloma patients include cardiovascular toxicity. Since carfilzomib's vascular effects are elusive, we investigated the vascular outcomes of carfilzomib and metformin (Met) coadministration. METHODS: Mice received: (i) saline; (ii) Cfz; (iii) Met; (iv) Cfz+Met for two consecutive (acute) or six alternate days (subacute protocol). Leucocyte-derived reactive oxygen species (ROS) and serum NOx levels were determined and aortas underwent vascular and molecular analyses. Mechanistic experiments were recapitulated in aged mice who received similar treatment to young animals. Primary murine (prmVSMCs) and aged human aortic smooth muscle cells (HAoSMCs) underwent Cfz, Met and Cfz+Met treatment and viability, metabolic flux and p53-LC3-B expression were measured. Experiments were recapitulated in AngII, CoCl2 and high-glucose stimulated HAoSMCs. RESULTS: Acutely, carfilzomib alone led to vascular hypo-contraction and increased ROS release. Subacutely, carfilzomib increased ROS release without vascular manifestations. Cfz+Met increased PGF2α-vasoconstriction and LC3-B-dependent autophagy in both young and aged mice. In vitro, Cfz+Met led to cytotoxicity and autophagy, while Met and Cfz+Met shifted cellular metabolism. CONCLUSION: Carfilzomib induces a transient vascular impairment and oxidative burst. Cfz+Met increased vascular contractility and synergistically induced autophagy in all settings. Therefore, carfilzomib cannot be accredited for a permanent vascular dysfunction, while Cfz+Met exert vasoprotective potency.

Preparation and characterization of a pterostilbene-peptide prodrug nanomedicine for the management of dry eye.

In the present study, a pterostilbene-peptide amphiphile (PS-GA-RGD) that can spontaneously self-assemble into prodrug nanomedicine, was rationally designed and developed as a novel ophthalmic formulation for the potential management of dry eye. The formed PS-GA-RGD nanomedicine was characterized by dynamic latter scattering (DLS) and transmission electron microscopy (TEM). After esterase treatment, active pterostilbene (PS) sustainably released from the PS-GA-RGD nanomedicine within 48 h, as indicated by an in vitro release study. In comparison with native PS, the formed PS-GA-RGD nanomedicine caused minimal cytotoxicity towards RAW 264.7 and HCEC cells in the 0-20 µM range and did not delay wound healing of HCEC monolayer within 6 h. Furthermore, PS-GA-RGD nanomedicine effectively reduced the intracellular reactive oxygen species (ROS) level in H2O2 challenged RAW264.7 macrophages and remarkably suppressed the secretion of inflammatory cytokines (e.g., NO, TNF-α, and IL-6) in lipopolysaccharide (LPS) activated RAW264.7 macrophages. Ocular tolerance to the proposed PS-GA-RGD nanomedicine was good after a single instillation in in vivo ocular irritation tests. Overall, the proposed PS-GA-RGD nanomedicine had potent anti-oxidant capacity and anti-inflammatory efficacy, which may be a promising ophthalmic formulation for the management of dry eye.

Preorganization Increases the Self-Assembling Ability and Antitumor Efficacy of Peptide Nanomedicine.

Inspired by the biological process of phosphorylation for which different sites of the same protein may have different activities and functions, we utilized phosphatase-based enzyme-instructed self-assembly (EISA) to construct self-assembled nanomedicine from the precursors with different phosphorylated sites. We found that, although the obtained self-assembling molecules after EISA were identical, the changes of EISA catalytic sites could determine the outcome of molecular self-assembly. The precursor with the phosphorylated site in the middle preorganized before EISA, while the ones with other phosphorylated sites could not preorganize before EISA. After EISA, the preorganized precursor then resulted in more stable and ordered assemblies than those of the others, which showed increased cellular uptake and up to 1.7-fold higher efficacy in an antitumor therapeutic compared to those assembled from unorganized precursors.

Comparing activity, toxicity and model membrane interactions of Jelleine-I and Trp/Arg analogs: analysis of peptide aggregation.

Increasing resistance in antibiotic and chemotherapeutic treatments has been pushing studies of design and evaluation of bioactive peptides. Designing relies on different approaches from minimalist sequences and endogenous peptides modifications to computational libraries. Evaluation relies on microbiological tests. Aiming a deeper understanding, we chose the octapeptide Jelleine-I (JI) for its selective and low toxicity profile, designed small modifications combining the substitutions of Phe by Trp and Lys/His by Arg and tested the antimicrobial and anticancer activity on melanoma cells. Biophysical methods identified environment-dependent modulation of aggregation, but critical aggregation concentrations of JI and analogs in buffer show that peptides start membrane interactions as monomers. The presence of model membranes increases or reduces the partial aggregation of peptides. Compared to JI, analog JIF2WR shows the lowest tendency to aggregation on bacterial model membranes. JI and analogs are lytic to model membranes. Their composition-dependent performance indicates preference for the higher charged anionic bilayers in line with their superior performance toward Staphylococcus aureus and Streptococcus pneumoniae. JIF2WR presented the higher partitioning, higher lytic activity and lower aggregated contents. Despite these increased membranolytic activities, JIF2WR exhibited comparable antimicrobial activity in relation to JI at the expenses of some loss in selectivity. We found that the substitution Phe/Trp (JIF2W) tends to decrease antimicrobial but to increase anticancer activity and aggregation on model membranes and the toxicity toward human cells. However, the concomitant substitution Lys/His by Arg (JIF2WR) modulates some of these tendencies, increasing both the antimicrobial and the anticancer activity while decreasing the aggregation tendency.

LMP2 Inhibitors as a Potential Treatment for Alzheimer's Disease.

The immunoproteasome (iP), an inducible proteasome variant harboring three immunosubunits, low molecular mass polypeptide-2 (LMP2), multicatalytic endopeptidase complex subunit-1, and low molecular mass polypeptide-7 (LMP7), is involved in multiple facets of inflammatory responses. We recently reported that YU102, a dual inhibitor of the iP subunit LMP2 and the constitutive proteasome catalytic subunit ß1, ameliorates cognitive impairments in mouse models of Alzheimer's disease (AD) independently of amyloid deposits. To investigate whether inhibition of LMP2 is sufficient to improve the cognitive functions of AD mice, here we prepared 37 YU102 analogues and identified a potent LMP2 inhibitor DB-310 (28) (IC50: 80.6 nM) with improved selectivity and permeability in cells overexpressing ABCB1 transporters. We show that DB-310 induces suppression of IL-1α production in microglia cells and improves cognitive functions in the Tg2576 transgenic mouse model of AD. This study supports that inhibition of LMP2 is a promising therapeutic strategy for treatment of AD.

Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe.

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.