An IL-7 fusion protein targeting EDA fibronectin upregulates TCF1 on CD8+ T-cells, preferentially accumulates to neoplastic lesions, and boosts PD-1 blockade.

BACKGROUND: Anti-PD-1 antibodies have revolutionized cancer immunotherapy due to their ability to induce long-lasting complete remissions in a proportion of patients. Current research efforts are attempting to identify biomarkers and suitable combination partners to predict or further improve the activity of immune checkpoint inhibitors. Antibody-cytokine fusions are a class of pharmaceuticals that showed the potential to boost the anticancer properties of other immunotherapies. Extradomain A-fibronectin (EDA-FN), which is expressed in most solid and hematological tumors but is virtually undetectable in healthy adult tissues, is an attractive target for the delivery of cytokine at the site of the disease. METHODS: In this work, we describe the generation and characterization of a novel interleukin-7-based fusion protein targeting EDA-FN termed F8(scDb)-IL7. The product consists of the F8 antibody specific to the alternatively spliced EDA of FN in the single-chain diabody (scDb) format fused to human IL-7. RESULTS: F8(scDb)-IL7 efficiently stimulates human peripheral blood mononuclear cells in vitro. Moreover, the product significantly increases the expression of T Cell Factor 1 (TCF-1) on CD8+T cells compared with an IL2-fusion protein. TCF-1 has emerged as a pivotal transcription factor that influences the durability and potency of immune responses against tumors. In preclinical cancer models, F8(scDb)-IL7 demonstrates potent single-agent activity and eradicates sarcoma lesions when combined with anti-PD-1. CONCLUSIONS: Our results provide the rationale to explore the combination of F8(scDb)-IL7 with anti-PD-1 antibodies for the treatment of patients with cancer.

Permutational Encoding Strategy Accelerates HIT Validation from Single-Stranded DNA-Encoded Libraries.

DNA-Encoded Libraries (DELs) allow the parallel screening of millions of compounds for various applications, including de novo discovery or affinity maturation campaigns. However, library construction and HIT resynthesis can be cumbersome, especially when library members present an unknown stereochemistry. We introduce a permutational encoding strategy suitable for the construction of highly pure single-stranded single-pharmacophore DELs, designed to distinguish isomers at the sequencing level (e.g., stereoisomers, regio-isomers, and peptide sequences). This approach was validated by synthesizing a mock 921,600-member 4-amino-proline single-stranded DEL ("DEL1"). While screening DEL1 against different targets, high-throughput sequencing results showed selective enrichment of the most potent stereoisomers, with enrichment factors that outperform conventional encoding strategies. The versatility of our methodology was additionally validated by encoding 24 scaffolds derived from different permutations of the amino acid sequence of a previously described cyclic peptide targeting Fibroblast Activation Protein (FAP-2286). The resulting library ("DEL2") was interrogated against human FAP, showing selective enrichment of five cyclic peptides. We observed a direct correlation between enrichment factors and on-DNA binding affinities. The presented encoding methodology accelerates drug discovery by facilitating library synthesis and streamlining HIT resynthesis while enhancing enrichment factors at the DEL sequencing level. This facilitates the identification of HIT candidates prior to medicinal chemistry and affinity maturation campaigns.

Targeted interleukin-2 enhances the in vivo anti-cancer activity of Pluvicto™.

PURPOSE: Pluvicto™ ([177Lu]Lu-PSMA-617), a radioligand therapeutic targeting prostate-specific membrane antigen (PSMA), has been recently approved for the treatment of metastatic castration-resistant prostate cancer (mCRPR). The drug suffers from salivary gland and kidney uptake that prevents its dose escalation to potentially curative doses. In this work, we sought to potentiate the in vivo anti-cancer activity of Pluvicto™ by combining it with L19-IL2, a clinical-stage investigational medicinal product based on tumor-targeted interleukin-2. METHODS: We established a new PSMA-expressing model (HT-1080.hPSMA) and validated it using a fluoresceine analogue of PSMA-617 (compound 1). The HT-1080.hPSMA model was used to study the saturation and tumor retention of Pluvicto™ (compound 2) and to run combination therapy studies with L19-IL2. To complement our understanding of the mechanism of action of this novel combination, we conducted proteomics experiments on tumor samples after therapy with Pluvicto™ alone or in combination with the immunocytokine. RESULTS: High, selective, and long-lived tumor uptake was observed for Pluvicto™ (2) in the novel HT-1080.hPSMA model. Therapy studies in HT-1080.hPSMA tumor-bearing mice revealed that the combination of Pluvicto™ (2) plus L19-IL2 mediated curative and durable responses in all animals. Potent in vivo anti-cancer activity was observed solely for the combination modality, at doses that were well tolerated by treated animals. Proteomics studies indicated that L19-IL2 boosts the activation of the immune system in animals pre-treated with Pluvicto™. CONCLUSION: The therapeutic efficacy of Pluvicto™ at low radioactive doses can be effectively enhanced by the combination with L19-IL2. Our findings warrant further clinical exploration of this novel combination modality.

A novel strategy to generate immunocytokines with activity-on-demand using small molecule inhibitors.

Cytokine-based therapeutics have been shown to mediate objective responses in certain tumor entities but suffer from insufficient selectivity, causing limiting toxicity which prevents dose escalation to therapeutically active regimens. The antibody-based delivery of cytokines significantly increases the therapeutic index of the corresponding payload but still suffers from side effects associated with peak concentrations of the product in blood upon intravenous administration. Here we devise a general strategy (named "Intra-Cork") to mask systemic cytokine activity without impacting anti-cancer efficacy. Our technology features the use of antibody-cytokine fusions, capable of selective localization at the neoplastic site, in combination with pathway-selective inhibitors of the cytokine signaling, which rapidly clear from the body. This strategy, exemplified with a tumor-targeted IL12 in combination with a JAK2 inhibitor, allowed to abrogate cytokine-driven toxicity without affecting therapeutic activity in a preclinical model of cancer. This approach is readily applicable in clinical practice.

In vivo activation of FAP-cleavable small molecule-drug conjugates for the targeted delivery of camptothecins and tubulin poisons to the tumor microenvironment.

Small molecule-drug conjugates (SMDCs) are increasingly considered as a therapeutic alternative to antibody-drug conjugates (ADCs) for cancer therapy. OncoFAP is an ultra-high affinity ligand of Fibroblast Activation Protein (FAP), a stromal tumor-associated antigen overexpressed in a wide variety of solid human malignancies. We have recently reported the development of non-internalizing OncoFAP-based SMDCs, which are activated by FAP thanks to selective proteolytic cleavage of the -GlyPro- linker with consequent release of monomethyl auristatin E (MMAE) in the tumor microenvironment. In this article, we describe the generation and the in vivo characterization of FAP-cleavable OncoFAP-drug conjugates based on potent topoisomerase I inhibitors (DXd, SN-38, and exatecan) and an anti-tubulin payload (MMAE), which are already exploited in clinical-stage and approved ADCs. The Glycine-Proline FAP-cleavable technology was directly benchmarked against linkers found in Adcetris™, Enhertu™, and Trodelvy™ structures by means of in vivo therapeutic experiments in mice bearing tumors with cellular or stromal FAP expression. OncoFAP-GlyPro-Exatecan and OncoFAP-GlyPro-MMAE emerged as the most efficacious anti-cancer therapeutics against FAP-positive cellular models. OncoFAP-GlyPro-MMAE exhibited a potent antitumor activity also against stromal models, and was therefore selected for clinical development.

Ex vivo mass spectrometry-based biodistribution analysis of an antibody-Resiquimod conjugate bearing a protease-cleavable and acid-labile linker.

Immune-stimulating antibody conjugates (ISACs) equipped with imidazoquinoline (IMD) payloads can stimulate endogenous immune cells to kill cancer cells, ultimately inducing long-lasting anticancer effects. A novel ISAC was designed, featuring the IMD Resiquimod (R848), a tumor-targeting antibody specific for Carbonic Anhydrase IX (CAIX) and the protease-cleavable Val-Cit-PABC linker. In vitro stability analysis showed not only R848 release in the presence of the protease Cathepsin B but also under acidic conditions. The ex vivo mass spectrometry-based biodistribution data confirmed the low stability of the linker-drug connection while highlighting the selective accumulation of the IgG in tumors and its long circulatory half-life.

Tumor-Targeted Interleukin 2 Boosts the Anticancer Activity of FAP-Directed Radioligand Therapeutics.

We studied the antitumor efficacy of a combination of 177Lu-labeled radioligand therapeutics targeting the fibroblast activation protein (FAP) (OncoFAP and BiOncoFAP) with the antibody-cytokine fusion protein L19-interleukin 2 (L19-IL2) providing targeted delivery of interleukin 2 to tumors. Methods: The biodistribution of 177Lu-OncoFAP and 177Lu-BiOncoFAP at different molar amounts (3 vs. 250 nmol/kg) of injected ligand was studied via SPECT/CT in mice bearing subcutaneous HT-1080.hFAP tumors, and self-absorbed tumor and organ doses were calculated. The in vivo anticancer effect of 5 MBq of the radiolabeled preparations was evaluated as monotherapy or in combination with L19-IL2 in subcutaneously implanted HT-1080.hFAP and SK-RC-52.hFAP tumors. Tumor samples from animals treated with 177Lu-BiOncoFAP, L19-IL2, or both were analyzed by mass spectrometry-based proteomics to identify therapeutic signatures on cellular and stromal markers of cancer and on immunomodulatory targets. Results: 177Lu-BiOncoFAP led to a significantly higher self-absorbed dose in FAP-positive tumors (0.293 ± 0.123 Gy/MBq) than did 177Lu-OncoFAP (0.157 ± 0.047 Gy/MBq, P = 0.01) and demonstrated favorable tumor-to-organ ratios at high molar amounts of injected ligand. Administration of L19-IL2 or 177Lu-BiOncoFAP as single agents led to cancer cures in only a limited number of treated animals. In 177Lu-BiOncoFAP-plus-L19-IL2 combination therapy, complete remissions were observed in all injected mice (7/7 complete remissions for the HT-1080.hFAP model, and 4/4 complete remissions for the SK-RC-52.hFAP model), suggesting therapeutic synergy. Proteomic studies revealed a mechanism of action based on the activation of natural killer cells, with a significant enhancement of the expression of granzymes and perforin 1 in the tumor microenvironment after combination treatment. Conclusion: The combination of OncoFAP-based radioligand therapeutics with concurrent targeting of interleukin 2 shows synergistic anticancer effects in the treatment of FAP-positive tumors. This experimental finding should be corroborated by future clinical studies.

A Comparative Analysis of Fibroblast Activation Protein-Targeted Small Molecule-Drug, Antibody-Drug, and Peptide-Drug Conjugates.

We present the first in vivo comparative evaluation of chemically defined antibody-drug conjugates (ADCs), small molecule-drug conjugates (SMDCs), and peptide-drug conjugates (PDCs) targeting and activated by fibroblast activation protein (FAP) in solid tumors. Both the SMDC (OncoFAP-Gly-Pro-MMAE) and the ADC (7NP2-Gly-Pro-MMAE) candidates delivered high amounts of active payload (i.e., MMAE) selectively at the tumor site, thus producing a potent antitumor activity in a preclinical cancer model.

An Antibody Targeting Fibroblast Activation Protein Simultaneously Fused to Interleukin-2 and Tumor Necrosis Factor Selectively Localizes to Neoplastic Lesions.

The delivery of specific cytokine payloads to a neoplastic environment employing antibodies able to selectively accumulate at the tumor site represents an attractive strategy to stimulate an immune response to cancer. Whilst conventional antibody-cytokine fusions based on a single payload have shown potent anticancer activity, the concomitant delivery of two cytokine payloads may further improve the therapeutic outcome as the immune system typically adopts multiple signals to reinforce an antitumor strategy. We here describe a potency-matched dual-cytokine antibody fusion protein containing a tumor-targeting antibody fragment specific to human fibroblast activation protein (FAP), simultaneously linked to both interleukin-2 (IL2) and a tumor necrosis factor (TNF) mutant. The resulting fusion protein, termed IL2-7NP2-TNFmut, formed stable non-covalent trimers driven by the interaction of the tumor necrosis factor subunits. Both cytokine payloads retained their biological activity within the fusion protein, as shown by in vitro cellular assays. The tumor-targeting properties and the anticancer activity of IL2-7NP2-TNFmut were investigated in vivo in immunocompromised mice bearing SKRC52 cells transduced with human FAP. The fusion protein preferentially localized to the cancer site and induced partial tumor retardation.

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties.

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1.

Fibroblast Activation Protein Triggers Release of Drug Payload from Non-internalizing Small Molecule Drug Conjugates in Solid Tumors.

PURPOSE: Small molecule drug conjugates (SMDC) are modular anticancer prodrugs that include a tumor-targeting small organic ligand, a cleavable linker, and a potent cytotoxic agent. Most of the SMDC products that have been developed for clinical applications target internalizing tumor-associated antigens on the surface of tumor cells. We have recently described a novel non-internalizing small organic ligand (named OncoFAP) of fibroblast activation protein (FAP), a tumor-associated antigen highly expressed in the stroma of most solid human malignancies. EXPERIMENTAL DESIGN: In this article, we describe a new series of OncoFAP-Drug derivatives based on monomethyl auristatin E (MMAE; a potent cytotoxic tubulin poison) and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment. RESULTS: The tumor-targeting potential of OncoFAP was confirmed in patients with cancer using nuclear medicine procedures. We used mass spectrometry methodologies to quantify the amount of prodrug delivered to tumors and normal organs, as well as the efficiency of the drug release process. Linkers previously exploited for anticancer drug conjugates were used as benchmark. We identified OncoFAP-Gly-Pro-MMAE as the best performing SMDC, which has now been prioritized for further clinical development. OncoFAP-Gly-Pro-MMAE selectively delivered more than 10% injected dose per gram of MMAE to FAP-positive tumors, with a tumor-to-kidney ratio of 16:1 at 24 hours post-injection. CONCLUSIONS: The FAP-specific drug conjugates described in this article promise to be efficacious for the targeting of human malignancies. The extracellular release of potent anticancer payloads mediates durable complete remission in difficult-to-treat animal models of cancer.

Mass Spectrometry-Based Method for the Determination of the Biodistribution of Tumor-Targeting Small Molecule-Metal Conjugates.

Nuclear medicine plays a key role in modern diagnosis and cancer therapy. The development of tumor-targeting radionuclide conjugates (also named small molecule-radio conjugates (SMRCs)) represents a significant improvement over the clinical use of metabolic radiotracers (e.g., [18F]-fluorodeoxyglucose) for imaging and over the application of biocidal external beam radiations for therapy. During the discovery of SMRCs, molecular candidates must be carefully evaluated typically by performing biodistribution assays in preclinical tumor models. Quantification methodologies based on radioactive counts are typically demanding due to safety concerns, availability of radioactive materials, and infrastructures. In this article, we report the development of a mass spectrometry (MS)-based method for the detection and quantification of small molecule-metal conjugates (SMMCs) as cold surrogates of SMRCs. We applied this methodology for the evaluation of the biodistribution of a particular class of tumor-targeting drug candidates based on natLu, natGa, and natF and directed against fibroblast activation protein (FAP). The reliability of the liquid chromatography-MS (LC-MS) analysis was validated by a direct comparison of MS-based and radioactivity-based biodistribution data. The results show that MS biodistribution of stable isotope metal conjugates is an orthogonal tool for the preclinical characterization of different classes of radiopharmaceuticals.

The ergogenic effect of acute carnosine and anserine supplementation: dosing, timing, and underlying mechanism.

Background: Recent studies suggest that acute-combined carnosine and anserine supplementation has the potential to improve the performance of certain cycling protocols. Yet, data on optimal dose, timing of ingestion, effective exercise range, and mode of action are lacking. Three studies were conducted to establish dosing and timing guidelines concerning carnosine and anserine intake and to unravel the mechanism underlying the ergogenic effects. Methods: First, a dose response study A was conducted in which 11 men randomly received placebo, 10, 20, or 30 mg.kg-1 of both carnosine and anserine. They performed 3x maximal voluntary isometric contractions (MVC), followed by a 5 x 6 s repeated cycling sprint ability test (RSA), once before the supplement and 30 and 60 minutes after. In a second study, 15 men performed 3x MVCs with femoral nerve electrical stimulation, followed by an RSA test, once before 30 mg.kg-1 carnosine and anserine and 60 minutes after. Finally, in study C, eight men performed a high intensity cycling training after randomly ingesting 30 mg.kg-1 of carnosine and anserine, a placebo or antihistamines (reduce post-exercise blood flow) to investigate effects on muscle perfusion. Results: Study A showed a 3% peak power (p = 0.0005; 95% CI = 0.07 to 0.27; ES = 0.91) and 4.5% peak torque (p = 0.0006; 95% CI = 0.12 to 0.50; ES = 0.87) improvement on RSA and MVC, with 30 mg.kg-1 carnosine + anserine ingestion 60 minutes before the performance yielding the best results. Study B found no performance improvement on group level; however, a negative correlation (r = -0.54; p = 0.0053; 95% CI = -0.77 to -0.19) was found between carnosinase enzyme activity (responsible for carnosine and anserine breakdown) and performance improvement. No effect of the supplement on neuromuscular function nor on muscle perfusion was found. Conclusions: These studies reveal that acute ingestion of 30 mg.kg-1 of both carnosine and anserine, 60 minutes before a high intensity exercise, can potentially improve performance, such as short cycling sprints or maximal muscle contractions. Subjects with lower carnosinase activity, and thus a slower breakdown of circulating dipeptides, appear to benefit more from this ergogenic effect. Finally, neither the involvement of a direct effect on neuromuscular function, nor an indirect effect on recovery through increased muscle perfusion could be confirmed as potential mechanism of action. The ergogenic mechanism therefore remains elusive.

Cyclo(His-Pro) Exerts Protective Carbonyl Quenching Effects through Its Open Histidine Containing Dipeptides.

Cyclo(His-Pro) (CHP) is a cyclic dipeptide which is endowed with favorable pharmacokinetic properties combined with a variety of biological activities. CHP is found in a number of protein-rich foods and dietary supplements. While being stable at physiological pH, CHP can open yielding two symmetric dipeptides (His-Pro, Pro-His), the formation of which might be particularly relevant from dietary CHP due to the gastric acidic environment. The antioxidant and protective CHP properties were repeatedly reported although the non-enzymatic mechanisms were scantly investigated. The CHP detoxifying activity towards α,ß unsaturated carbonyls was never investigated in detail, although its open dipeptides might be effective as already observed for histidine containing dipeptides. Hence, this study investigated the scavenging properties of TRH, CHP and its open derivatives towards 4-hydroxy-2-nonenal. The obtained results revealed that Pro-His possesses a marked activity and is more reactive than l-carnosine. As investigated by DFT calculations, the enhanced reactivity can be ascribed to the greater electrophilicity of the involved iminium intermediate. These findings emphasize that the primary amine (as seen in l-carnosine) can be replaced by secondary amines with beneficial effects on the quenching mechanisms. Serum stability of the tested peptides was also evaluated, showing that Pro-His is characterized by a greater stability than l-carnosine. Docking simulations suggested that its hydrolysis can be catalyzed by serum carnosinase. Altogether, the reported results evidence that the antioxidant CHP properties can be also due to the detoxifying activity of its open dipeptides, which might be thus responsible for the beneficial effects induced by CHP containing food.

Characterization of Catecholaldehyde Adducts with Carnosine and l-Cysteine Reveals Their Potential as Biomarkers of Catecholaminergic Stress.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of dopamine and norepinephrine to produce 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), respectively. Both of these aldehydes are potently cytotoxic and have been implicated in pathogenesis of neurodegenerative and cardiometabolic disorders. Previous work has demonstrated that both the catechol and aldehyde moieties of DOPAL are reactive and cytotoxic via their propensity to cause macromolecular cross-linking. With certain amines, DOPAL likely reacts via a Schiff base before oxidative activation of the catechol and rearrangement to a stable indole product. Our current work expands on this reactivity and includes the less-studied DOPEGAL. Although we confirmed that antioxidants mediated DOPAL's reactivity with carnosine and N-acetyl-l-lysine, antioxidants had no effect on reactivity with l-cysteine. Therefore, we propose a non-oxidative mechanism where, following Schiff base formation, the thiol of l-cysteine reacts to form a thiazolidine. Similarly, we demonstrate that DOPEGAL forms a putative thiazolidine conjugate with l-cysteine. We identified and characterized both l-cysteine conjugates via HPLC-MS and additionally identified a DOPEGAL adduct with carnosine, which is likely an Amadori product. Furthermore, we were able to demonstrate that these conjugates are produced in biological systems via MAO after treatment of the cell lysate with norepinephrine or dopamine along with the corresponding nucleophiles (i.e., l-cysteine and carnosine). As it has been established that metabolic and oxidative stress leads to increased MAO activity and accumulation of DOPAL and DOPEGAL, it is conceivable that conjugation of these aldehydes to carnosine or l-cysteine is a newly identified detoxification pathway. Furthermore, the ability to characterize these adducts via analytical techniques reveals their potential for use as biomarkers of dopamine or norepinephrine metabolic disruption.

Synthesis and characterization of 13C labeled carnosine derivatives for isotope dilution mass spectrometry measurements in biological matrices.

Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the 13C-labeled carnosine analogs and their application as internal standards for the quantitation of carnosine and anserine in a biological matrix.

PHoral: Effects of carnosine supplementation on quantity/quality of oral salivae in healthy volunteer and in subjects affected by common oral pathologies.

BACKGROUND: Diseases of the oral cavity (OC) with an infectious trigger such as caries and periodontal disease are extremely common in the general population and can also have effects at the cardiovascular level. The oral salivary flow, with its buffering capacity, is able to regulate the pH of the OC and, therefore, significantly contribute to the ecological balance of the microenvironment in which the oral microbiome (OM) develops. On the other side, when the quality/quantity of salivary flow is altered it is supposed the disruption of this balance with the potential increase in oral pathogens and triggered diseases. Among the endogenous substances able to exert a significant effect on the salivary flow and its characteristics, carnosine (Car), a dipeptide originally isolated in skeletal muscle, represents, thanks to the known buffering properties, a promising principle. METHODS: We aimed this protocol to evaluate the quantitative/qualitative characteristics of the salivary flow in healthy volunteer subjects (n = 20) and in subjects suffering from common OC pathologies (n = 40), before and after 7 days of supplementation with SaliflussTM (Metis Healthcare srl, Milan, Italy), a Class I medical device on the market as 400 mg mucoadhesive oral tablets that has Car as the main ingredient. DISCUSSION: Combining the characteristics of saliva with the OM and comparing them with OC pathologies, we expect to clarify their reciprocal relationship and, using quantitative proteomics techniques, to help clarify the mechanism of action of Car.

Understanding the antioxidant and carbonyl sequestering activity of carnosine: direct and indirect mechanisms.

Carnosine is an endogenous dipeptide whose oral administration has been found to prevent several oxidative based diseases including lung disease, type 2 diabetes and its micro and macrovascular complications, cardiovascular disorders, neurodegenerative and kidney disease. While it is generally accepted that the beneficial effects of carnosine are due to its antioxidant, anti-advanced glycation end product (AGE) and -advanced lipoxidation end product (ALE) and anti-inflammatory properties, the molecular mechanisms explaining such effects have not yet been clearly defined. Studies indicate that carnosine acts by a direct antioxidant mechanism and by sequestering reactive carbonyls (RCS), the byproducts of lipid and glucose oxidation, thus inhibiting AGE and ALE which are the reaction products of RCS with proteins. Moreover, carnosine has also been found to act indirectly by activating the Nrf2 transcription factor, a mechanism that would explain many of the effects evoked by this peptide such as anti-inflammatory, antioxidant, antiglycation and anti-carbonyl effects and taken together would explain its therapeutic effect. The present review reports and discusses the most recent studies on the molecular mechanisms of carnosine which need to be fully clarified before promoting carnosine and derivatives as therapeutic agents.

Towards the Inhibition of Protein-Protein Interactions (PPIs) in STAT3: Insights into a New Class of Benzothiadiazole Derivatives.

Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target due to the relationship between its constitutive activation and malignant tumors. Through a virtual screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide (1) was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based assay. Among them, benzosulfamide 1 showed a significant activity with an IC50 = 15.8 ± 0.6 µM as a direct STAT3 inhibitor. Notably, we discovered that compound 1 was also able to interact with cysteine residues located around the SH2 domain. By applying mass spectrometry, liquid chromatography, NMR, and UV spectroscopy, an in-depth investigation was carried out, shedding light on its intriguing and unexpected mechanism of interaction.

Development of a direct LC-ESI-MS method for the measurement of human serum carnosinase activity.

Carnosine (ß-alanyl-L-histidine) is a natural peptide that have been described as a potential pharmacological agent owing to some positive outcomes from several pharmacological tests in animal models of human diseases. However, carnosine has limited activity in humans since the peptide upon absorption is rapidly hydrolyzed in the serum by the enzyme carnosinase (i.e. CN1; E.C. 3.4.13.20). Over the years the main approaches aimed at limiting carnosine hydrolysis have been focused on obtaining CN1-stable derivatives with an increased bioavailability and unmodified or enhanced activity. Only recently the hypothesis of co-administration of carnosine and selective inhibitors of CN1 have been proposed. Such an approach requires reliable methods for screening the effect on carnosine hydrolysis rate operated by CN1 in a throughput scale allowing to test from few compounds up to whole compound libraries. The only assay with such features available in literature relies on ortho-phtalaldehyde (OPA) derivatization of the hydrolysis product (i.e. histidine), followed by a fluorimetric read. Herein, we propose an alternative method based on a direct measurement of the residual substrate by liquid chromatography-mass spectrometry (LC-MS). The assay demonstrated to be reliable since gave results comparable to literature data concerning the hydrolysis rate of carnosine as determined into human serum. Moreover, the method was quite flexible and easily adaptable to a substrate change, as demonstrated by the measurement of the hydrolysis rate of all the natural analogs of carnosine. In this context the data collected for anserine suggest that our method looked more reliable and substrate change can undergo an underestimation of hydrolytic activity in OPA -based assays.