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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.

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.

Supplementary Information

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.

Reactive Carbonyl Species are electrophiles generated by the oxidative cleavage of lipids and sugars. Such compounds have been described as important molecules for cellular signaling, whilst their accumulation has been found to be cytotoxic as they may trigger aberrant modifications of proteins (a process often referred to as carbonylation).:A correlation between carbonylation of proteins and human disease progression has been shown in ageing, diabetes, obesity, chronic renal failure, neurodegeneration and cardiovascular disease. However, the fate of reactive carbonyl species is still far from being understood, especially concerning the mechanisms responsible for their disposal as well as the importance of this in disease progression.:In this context, some data have been published on phase I and phase II deactivation of reactive carbonyl species. In the case of phase II mechanisms, the route involving glutathione conjugation and subsequent disposal of the adducts has been extensively studied both in vitro and in vivo for some of the more representative compounds, e.g. 4-hydroxynonenal.:There is also emerging evidence of an involvement of carnosine as an endogenous alternative to glutathione for phase II conjugation. However, the fate of carnosine conjugates is still poorly investigated and, unlike glutathione, there is little evidence of the formation of carnosine adducts in vivo. The acquisition of such data could be of importance for the development of new drugs, since carnosine and its derivatives have been proposed as potential therapeutic agents for the mitigation of carbonylation associated with disease progression.:Herein, we wish to review our current knowledge of the binding of reactive carbonyl species with carnosine together with the disposal of carnosine conjugates, emphasizing those aspects still requiring investigation such as conjugation reversibility and enzyme assisted catalysis of the reactions.

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,β 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.

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 material, 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, natF and directed against Fibroblast Activation Protein (FAP). The reliability of the LC-MS analysis was validated by direct comparison of MSbased and radioactivity-based biodistribution data. Results show that MS biodistribution of stable isotope metal conjugates is an orthogonal tool for the preclinical characterization of different classes of radiopharmaceuticals.

The use of cytokine-based therapeutics has traditionally been limited by systemic toxicity, but it is now becoming evident that it may be possible to circumvent this problem in a number of ways. Tumor-targeting antibody-cytokine fusion proteins (also called “immunocytokines”) typically allow dose escalation to therapeutically active regimens as a result of their selective localization at the site of disease (Neri 2019; Helguera, Morrison, and Penichet 2002). Despite the enhancement of the therapeutic index, when the concentration of the payload in blood exceeds a certain threshold, patients may experience dose-limiting immune-related adverse events (irAEs). In most cases, irAEs disappear when the immunocytokine is cleared from circulation (Ko et al. 2004; Spitaleri et al. 2013; Rudman et al. 2011). Here, we describe an innovative approach to mask off-target activity of immunocytokines by transient inhibition of their intracellular signaling cascade. Small molecules are particularly attractive for this application since their serum half-life matches the clearance rate of the immunocytokine from circulation. L19-TNF and L19-IL12 are two clinical-stage antibody-cytokine fusion proteins that display potent activity by triggering the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Janus Kinase 2 (JAK2) signaling pathways, respectively (Amin et al. 2018; Hu et al. 2021). In a first study, GSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice with the aim to reduce acute systemic toxicity associated with TNF signaling. Transient inhibition of RIPK1 allowed the administration of L19-murTNF doses which would otherwise be lethal. The combination with GSK’963 did not affect the selective localization of the immunocytokine to tumors. Moreover, L19-murTNF was still able to induce tumor necrosis and to exert potent anti-cancer activity. In a second study, tumor-bearing mice receiving L19-murIL12 were pretreated with Ruxolitinib, a commercially available JAK2 inhibitor. The addition of Ruxolitinib could significantly improve the tolerability profile of L19-murIL12 without affecting the anti-cancer properties of the immunocytokine. In clinical trials, it has been observed that IL12 treatment can induce cytokine release syndrome and liver damage (Atkins et al. 1997). Our data demonstrate that the pre-treatment with Ruxolitinib restricted the pro-inflammatory effects of L19-murIL12 to the tumor site, protecting the mice from body weight loss as well as reducing blood cytokine levels and hepatotoxicity. Overall, this preclinical work is of clinical relevance, as patients treated with targeted cytokines could potentially benefit from judicious combinations treatments using small molecule inhibitors. Citation Format: Giulia Rotta, Ettore Gilardoni, Domenico Ravazza, Jacqueline Mock, Samuele Cazzamalli, Roberto De Luca, Emanuele Puca, Dario Neri, Sheila Dakhel Plaza. Decreasing toxicity of immunocytokines by transient and selective inhibition of their intracellular signaling activation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5744.

Conventional cancer chemotherapy relies on the use of cytotoxic drugs which are not designed and capable of selective accumulation to neoplastic lesions. The conjugation to tumour-targeting delivery vehicles has been proposed as a strategy to enhance the therapeutic index of potent cytotoxic drugs. To date, fifteen Antibody-Drug Conjugate products have received marketing approval for the treatment of solid and liquid human malignancies. We present an in vivo comparative evaluation of ADCs against products based on small organic delivery vehicles (Small Molecule-Drug Conjugates, or SMDCs) or on tumour-targeting peptides (Peptide-Drug Conjugates, or PDCs). All products are designed to target and to be activated by Fibroblast Activation Protein in the tumour microenvironment. Our results reveal that both targeted ADC and SMDC products mediate potent anti-cancer activity and complete tumour remission in a FAP-positive preclinical murine model of cancer, when administered at the same molar dose. Non-targeted albumin and antibody conjugates included in our comparative studies delayed tumour growth but presented suboptimal anti-cancer efficacy in the same cancer model. Our results indicate that both FAP-targeted ADCs and SMDCs are promising product candidates for the treatment of multiple human malignancies. Citation Format: Aureliano Zana, Claudia Puig Moreno, Ettore Gilardoni, Dario Neri, Samuele Cazzamalli. FAP-targeted non-internalising drug conjugates: A comparative evaluation between small molecules, antibodies and peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1988.

Radioligand Therapeutics (RLTs) are products composed of a tumor-targeting small organic ligand and a biocidal radioactive payload. The therapeutic potential of RLTs strongly depends on their uptake and residence time in tumors and in the healthy structures after administration. Fibroblast Activation Protein (FAP) is a cell surface antigen abundantly expressed in the microenvironment of the majority of solid human cancers of epithelial origin. We have recently described OncoFAP, an ultra-high affinity ligand of FAP whose targeting performances have been validated by Nuclear Medicine in patients with a wide variety of solid malignancies. Multivalent OncoFAP radioligand therapeutics (OncoFAP-RLTs) show high and prolonged tumor uptake with favorable tumor-to-organ ratios. Here we describe the development of novel multivalent OncoFAP-RLTs and their combination with a clinical-stage tumor-targeted interleukin-2 (L19-IL2). We designed and synthesized a set of multivalent OncoFAP derivatives, aiming at maximizing their affinity for the target protein and their residence time in the catalytic pocket of FAP. The multivalent ligands were coupled to the DOTAGA chelator, suitable for radiolabeling with 177Lu for therapeutic application. The tumor targeting properties of the multivalent OncoFAP ligands were investigated in a comparative biodistribution study in SK-RC-52.hFAP tumor-bearing mice. 177Lu-OncoFAP-23 emerged for its superior uptake and residence time in tumors and for its favorable tumor-to-kidney ratio. Moreover, when administered at very low dose 177Lu-OncoFAP-23 exhibited an enhanced anticancer activity compared to a monovalent (177Lu-OncoFAP-DOTAGA) and bivalent (177Lu-BiOncoFAP-DOTAGA) version of OncoFAP. When OncoFAP-based RLTs were combined with L19-IL2, a strong synergistic anticancer activity was observed. This synergy is driven by NK-cell mediated immune response directed to the tumor, with the concomitant release of granzymes and other pro-apoptotic proteins in the tumor microenvironment. Our findings heighten OncoFAP-based RLTs as promising anti-cancer drugs for treatment of FAP-positive tumors, alone or in combination with targeted interleukin-2. Citation Format: Andrea Galbiati, Matilde Bocci, Ettore Gilardoni, Aureliano Zana, Dario Neri, Samuele Cazzamalli. Tumor-targeted interleukin-2 enhances the anti-cancer activity of multivalent OncoFAP radioligand therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2838.

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.

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

Small Molecule-Drug Conjugates (SMDCs) are modular anti-cancer pro-drugs that include a tumor-targeting small ligand, a cleavable linker and a potent cytotoxic agent. SMDC products that have been developed for clinical applications are targeting internalizing tumor-associated antigens expressed on the surface of tumor cells. We have recently developed 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 of solid human malignancies. The tumor targeting performance of OncoFAP has been validated by nuclear medicine studies in patients with various solid tumors. In a previous study, we showed that OncoFAP can be used to produce non-internalizing SMDCs that are effective and well tolerated. Here, we describe a new series of OncoFAP-Drug derivatives based on the MMAE tubulin poison and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment. We benchmarked the new SMDCs against OncoFAP-MMAE conjugates displaying linker modules which are widely used in approved and clinical stage Antibody-Drug Conjugates, including structures cleaved by Cathepsin B and by reducing agents. We selected OncoFAP-GlyPro-MMAE as the most efficacious and safe SMDC for further clinical development after quantitatively analyzing the biodistribution of MMAE released by OncoFAP-MMAE conjugates. OncoFAP-GlyPro-MMAE selectively delivers high amounts of MMAE at the site of disease, with a tumor-to-kidney ratio of 7-to-1 and of 16-to-1 at 6- and 24-hours post-injection, respectively. Our molecules based on the OncoFAP tumor-targeting ligand and FAP-cleavable linkers promise to be safe and effective against most of human malignancies. Citation Format: Aureliano Zana, Andrea Galbiati, Ettore Gilardoni, Jacopo Millul, Theo Sturm, Riccardo Stucchi, Matilde Bocci, Abdullah Elsayed, Lisa Nadal, Martina Cirillo, Dario Neri, Samuele Cazzamalli. Fibroblast activation protein triggers the release of drug payload from non-internalizing small molecule-drug conjugates in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB522.

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.

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward α,β-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine.

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.

Carnosine (β-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.

Carnosine (beta-alanyl-L-histidine) and its methylated analogue anserine are present in relevant concentrations in the omnivore human diet. Several studies reported promising therapeutic potential for carnosine in various rodent models of oxidative stress and inflammation-related chronic diseases. Nevertheless, the poor serum stability of carnosine in humans makes the translation of rodent models hard. Even though anserine and carnosine have similar biochemical properties, anserine has better serum stability. Despite this interesting profile, the research on anserine is scarce. The aim of this study was to explore the bioavailability and stability of synthesized anserine by (1) performing in vitro stability experiments in human plasma and molecular modelling studies and by (2) evaluating the plasma and urinary pharmacokinetic profile in healthy volunteers following different doses of anserine (4-10-20 mg/kg body weight). A bio-analytical method for measuring anserine levels was developed and validated using liquid chromatography-electrospray mass spectrometry. Both plasma (CMAX: 0.54-1.10-3.12 µM) and urinary (CMAX: 0.09-0.41-0.72 mg/mg creatinine) anserine increased dose-dependently following ingestion of 4-10-20 anserine mg/kg BW, respectively. The inter-individual variation in plasma anserine was mainly explained by the activity (R2 = 0.75) and content (R2 = 0.77) of the enzyme serum carnosinase-1. Compared to carnosine, a lower interaction energy of anserine with carnosinase-1 was suggested by molecular modelling studies. Conversely, the two dipeptides seems to have similar interaction with the PEPT1 transporter. It can be concluded that nutritionally relevant doses of synthesized anserine are well-absorbed and that its degradation by serum carnosinase-1 is less pronounced compared to carnosine. This makes anserine a good candidate as a more stable carnosine-analogue to attenuate chronic diseases in humans.

Carnosine is present in high concentrations in specific human tissues such as the skeletal muscle, and among its biological functions, the remarkable scavenging activity toward reactive carbonyl species is noteworthy. Although the two enantiomers show almost identical scavenging reactivity toward reactive carbonyl species, only d-carnosine is poorly adsorbed at the gastrointestinal level and is stable in human plasma. Direct methods for the enantioselective analysis of carnosine are still missing even though they could find more effective applications in the analysis of complex matrices. In the present study, the use of two different chiral stationary phases is presented. A chiral ligand-exchange chromatography stationary phase based on N,S-dioctyl-d-penicillamine resulted in the direct enantioseparation of carnosine. Indeed, running the analysis at 25°C and 1.0 mL/min with a 1.5 mM copper(II) sulfate concentration allowed us to obtain separation and resolution factors of 3.37 and 12.34, respectively. However, the use of a copper(II)-containing eluent renders it hardly compatible with mass spectrometry detectors. With the teicoplanin-based stationary phase, a mass spectrometry compatible method was successfully developed. Indeed, a water/methanol 60:40 v/v pH 3.1 eluent flowed at 1.0 mL/min and with a 25°C column temperature produced separation and resolution factors of 2.60 and 4.16, respectively.

The study combines HPLC-based with MS-based competitive analyses to evaluate the quenching activity of a set of carnosine derivatives towards methylglyoxal (MGO) and malondialdehyde (MDA) chosen as representative of α- and β-dicarbonyls, respectively. The obtained results underline that these derivatives are moderately reactive towards MDA with which they form the corresponding N-propenal adduct via Michael addition. In contrast they proved a rather poor quenching activity towards MGO with which they can condense to give MOLD-like adducts through a concerted mechanism involving more quenchers molecules. Even though both quenching mechanisms involve the amino group in its neutral form, in silico studies revealed that the reported reactivity values depend on different stereo-electronic parameters which are reflected in the different observed quenching mechanism. Finally, the MGO quenching reactivity and the unselective (and unwanted) pyridoxal quenching are found to be influenced by the same parameters thus rationalizing the known difficulty in the design of potent and selective quenchers towards β-dicarbonyls.

The chiral purity of some molecules such as nutraceuticals is fundamental to ensure their beneficial activities and it must be checked during quality control analysis. Carnosine is a natural histidine dipeptide used as ingredient for food supplements, but only his L-enantiomer is absorbed and active. Despite of this feature, a method for the separation of carnosine enantiomers without derivatization has only recently been published. Herein, we validated a method based on a Chirobiotic T column and an UV detector for the direct quantification of carnosine enantiomers, following ICH guideline. Moreover, we demonstrated that elution with water containing 0.1% formic acid and 20–40% ensures stereo-, chemo- and regio-selectivity for the separation and the identification of carnosine enantiomers and natural analogues. Moreover, the method allows a direct hyphenation with electrospray mass spectrometry to increase detection selectivity and sensitivity. As far as we know, this is the first method allowing the simultaneous identification and quantification of natural analogues of carnosine, which can be important for application such as the identification of enantiomeric impurities or adulteration that can occur during the storage or the preparation of foods or food supplements containing histidine dipeptides.

Cardiovascular disease is a severe health problem, especially in the Western world, and its primary cause is atherosclerosis, which is characterized by the arterial wall thickening. Modern therapeutic strategies have restricted efficacy and mortality still remains high[1]. Current research has supported the idea of targeting disregulated endothelial cell (EC) metabolism as a novel therapeutic strategy[2]. In the scope of MSCA Moglynet EJD, we aim to further explore the possibilities for an improved treatment of this life threatening disease. EC glycolytic flux is up-regulated during angiogenesis and it is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3)[3], which is hence an innovative target for atherosclerosis therapy. PFKFB3 is a homodimeric bifunctional enzyme that has a very high kinase to phosphatase activity ratio. Its activity is controlled by the N-terminus autoregulatory domain in the kinase region[4]. Virtual screening was performed on the targeted allosteric binding site and here we present the synthesis and biological evaluation of the selected libraries of PFKFB3 phosphatase modulators deriving from two design strategies. In vitro activity and binding assays were performed on the isolated recombinant enzyme. Phosphatase activity method was developed in-house using LC-MS instrument and binding assay was performed using Microscale thermophoresis. [1] J. Goveia, P. Stapor, P. Carmeliet, EMBO Mol Med 2016, 6, 1105–1120. [2] D. J. Rader, A. Daugherty, Nature 2008, 541, 904-913. [3] J. Chesney,R. Mitchell,F. Benigni,M. Bacher,L. Spiegel,Y. Al-Abed,J.H. Han,C. Metz, R. Bucala, Proc Natl Acad Sci U S A 1999, 96, 3047–3052. [4] S.G. Kim, N.P. Manes, M.R. El-Maghrabi, Y.H. Lee, J.Biol.Chem 2006, 281, 2939-2944.

The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods.