A simplified genomic profiling approach predicts outcome in metastatic colorectal cancer

The response of metastatic colorectal cancer (mCRC) to the first-line conventional combination therapy is highly variable, reflecting the elevated heterogeneity of the disease. The genetic alterations underlying this heterogeneity have been thoroughly characterized through omic approaches requiring elevated efforts and costs. In order to translate the knowledge of CRC molecular heterogeneity into a practical clinical approach, we utilized a simplified Next Generation Sequencing (NGS) based platform to screen a cohort of 77 patients treated with first-line conventional therapy. Samples were sequenced using a panel of hotspots and targeted regions of 22 genes commonly involved in CRC. This revealed 51 patients carrying actionable gene mutations, 22 of which carried druggable alterations. These mutations were frequently associated with additional genetic alterations. To take into account this molecular complexity and assisted by an unbiased bioinformatic analysis, we defined three subgroups of patients carrying distinct molecular patterns. We demonstrated these three molecular subgroups are associated with a different response to first-line conventional combination therapies. The best outcome was achieved in patients exclusively carrying mutations on TP53 and/or RAS genes. By contrast, in patients carrying mutations in any of the other genes, alone or associated with mutations of TP53/RAS, the expected response is much worse compared to patients with exclusive TP53/RAS mutations. Additionally, our data indicate that the standard approach has limited efficacy in patients without any mutations in the genes included in the panel. In conclusion, we identified a reliable and easy-to-use approach for a simplified molecular-based stratification of mCRC patients that predicts the efficacy of the first-line conventional combination therapy

The Energy Landscape of Human Serine Racemase

Human serine racemase is a pyridoxal 5′-phosphate (PLP)-dependent dimeric enzyme that catalyzes the reversible racemization of L-serine and D-serine and their dehydration to pyruvate and ammonia. As D-serine is the co-agonist of the N-methyl-D-aspartate receptors for glutamate, the most abundant excitatory neurotransmitter in the brain, the structure, dynamics, function, regulation and cellular localization of serine racemase have been investigated in detail. Serine racemase belongs to the fold-type II of the PLP-dependent enzyme family and structural models from several orthologs are available. The comparison of structures of serine racemase co-crystallized with or without ligands indicates the presence of at least one open and one closed conformation, suggesting that conformational flexibility plays a relevant role in enzyme regulation. ATP, Mg2+, Ca2+, anions, NADH and protein interactors, as well as the post-translational modifications nitrosylation and phosphorylation, finely tune the racemase and dehydratase activities and their relative reaction rates. Further information on serine racemase structure and dynamics resulted from the search for inhibitors with potential therapeutic applications. The cumulative knowledge on human serine racemase allowed obtaining insights into its conformational landscape and into the mechanisms of cross-talk between the effector binding sites and the active site

Caratterizzazione biochimica di serina racemasi umana: substrati, inibitori ed effettori allosterici

In recent years, many neurodegenerative diseases have been correlated to the dysfunction of N-methyl-D-aspartate receptors (NMDARs)-mediated neurotransmission. Drugs that directly affect the activity of the receptors evoke strong responses and typically show severe side effects. In this framework, serine racemase (SR) – the enzyme responsible for D-serine production in the brain – was suggested as an alternative therapeutic target. The detailed characterization of human SR (hSR) is currently rather limited, with a few published biochemical studies that provides a limited insight into the understanding of its regulation mechanisms. This work was aimed at achieving a more complete characterization of hSR dynamics, function and regulatory properties. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, the dependence of L- and D-serine β-elimination and L-serine racemization activities on ATP concentration was characterized and found to be strongly cooperative. ATP binding to the holo-enzyme, monitored by the fluorescence changes of the coenzyme, was also determined to be cooperative. The well-known active site ligands glycine and malonate increase the hSR affinity for ATP and, conversely, ATP increases the non-cooperative glycine and malonate binding. These results indicated a cross-talk between allosteric and active sites, leading to the stabilization of two alternative protein conformations endowed with significantly different microscopic dissociation constants for ATP , as estimated by applying the Monod, Wyman and Changeux model. To further investigate this allosteric communication, we probed the active site accessibility by quenching of the coenzyme fluorescence in the absence and presence of ATP. We found that ATP stabilized a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation. We also investigated the effect of halides on hSR activity and we found that it is affected in the order fluoride>chloride>bromide. On the contrary, iodide elicited a complete inhibition, accompanied by a modulation of the tautomeric equilibrium of the internal aldimine. Finally, we applied two different approaches for the search of new hSR competitive inhibitors. With the first approach, we developed a new series of compounds based on cyclopropane scaffold and on malonate core modifications. With the second approach, a library of compound was screened against hSR structure. The positive molecules were tested with in vitro analysis. Our results can provide useful indications for the development of novel SR inhibitors.Negli ultimi anni è stato accertato che molte malattie neurodegenerative risultano correlate a disfunzioni della trasmissione neuro-eccitatoria mediata dai recettori per il N-metil-D-aspartato (NMDAR). I farmaci che regolano direttamente l’attività di tali recettori causano l’insorgenza di gravi effetti collaterali. A questo proposito la serina racemasi (SR) – l’enzima responsabile della sintesi della D-serina a livello cerebrale – può rappresentare un alternativo bersaglio terapeutico. Attualmente la caratterizzazione dettagliata della SR umana (hSR) è ancora limitata, con la pubblicazione di pochi studi biochimici che restituiscono una visione completa per la comprensione dei suoi meccanismi di regolazione. Questo lavoro è stato volto all’ottenimento di una più completa caratterizzazione delle dinamiche, della funzione e delle proprietà regolative di hSR. Dopo aver migliorato le rese di purificazione e la stabilità della proteina, che finora avevano limitato la caraterizzazione biochimica di hSR, è stata caratterizzata la dipendenza dall’ATP dell’attività di β-eliminazione della L- e D-serina e di racemizzazione della L-serina ed è risultata essere fortemente cooperativa. Il legame dell’ATP all’oloenzima, monitorato per mezzo delle variazioni di fluorescenza del coenzima, è risultato a sua volta cooperativo. I ligandi noti del sito attivo glicina e malonato aumentano l’affinità di hSR per l’ATP e, d’altra parte, l’ATP aumenta l’affinità di legame non cooperativo di glicina e malonato. Questi risultati indicano una comunicazione reciproca tra il sito attivo e il sito allosterico, portando alla stabilizzazione di due conformazioni alternative che presentano costanti di dissociazione microscopiche significativamente differenti, come stimato tramite l’analisi con il modello di Monod, Wyman and Changeux. Per comprendere ulteriormente questa comunicazione allosterica, abbiamo verificato l’accessibilità del sito attivo attraverso il quenching della fluorescenza del coenzima, in presenza e in assenza di ATP. È stato verificato che l’ATP stabilizza una conformazione chiusa della sito attivo in aldimina esterna, suggerendo un possibile meccanismo per l’attivazione dell’hSR indotta dall’ATP. Abbiamo inoltre studiato l’effetto degli alogenuri sull’attività di hSR, con un influenza nell’ordine fluoruro>cloruro>bromuro. Al contrario, lo ioduro promuove una completa inibizione, accompagnata da una modulazione dell’equilibrio tautomerico dell’aldimina interna. In conclusione, abbiamo applicato due diversi approcci per la ricerca di nuovi inibitori competitive dell’hSR. Nel primo caso abbiamo sviluppato una serie di composti basati sullo scaffold del ciclopropano e sulla modificazione del nucleo del malonato. Il secondo approccio ha previsto lo screening di una libreria di composti contro la struttura dell’enzima. Le molecole positive sono state poi testate in vitro. Questi risultati possono fornire utili indicazioni per lo sviluppo di inibitori innovativi per hSR

Enzyme Replacement Therapy for Genetic Disorders Associated with Enzyme Deficiency

Mutations in human genes might lead to loss of functional proteins, causing diseases. Among these genetic disorders, a large class is associated with the deficiency in metabolic enzymes, resulting in both an increase in the concentration of substrates and a loss in the metabolites produced by the catalyzed reactions. The identification of therapeutic actions based on small molecules represents a challenge to medicinal chemists because the target is missing. Alternative approaches are biology-based, ranging from gene and stem cell therapy, CRISPR/Cas9 technology, distinct types of RNAs, and enzyme replacement therapy (ERT). This review will focus on the latter approach that since the 1990s has been successfully applied to cure many rare diseases, most of them being lysosomal storage diseases or metabolic diseases. So far, a dozen enzymes have been approved by FDA/EMA for lysosome storage disorders and only a few for metabolic diseases. Enzymes for replacement therapy are mainly produced in mammalian cells and some in plant cells and yeasts and are further processed to obtain active, highly bioavailable, less degradable products. Issues still under investigation for the increase in ERT efficacy are the optimization of enzymes interaction with cell membrane and internalization, the reduction in immunogenicity, and the overcoming of blood-brain barrier limitations when neuronal cells need to be targeted. Overall, ERT has demonstrated its efficacy and safety in the treatment of many genetic rare diseases, both saving newborn lives and improving patients' life quality, and represents a very successful example of targeted biologics

Quenching of tryptophan fluorescence in a highly scattering solution: Insights on protein localization in a lung surfactant formulation.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic surfactant developed for respiratory distress syndrome replacement therapy in pre-term newborn infants. CHF5633 contains two phospholipids (dipalmitoylphosphatidylcholine and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol sodium salt), and peptide analogues of surfactant protein C (SP-C analogue) and surfactant protein B (SP-B analogue). Both proteins are fundamental for an optimal surfactant activity in vivo and SP-B genetic deficiency causes lethal respiratory failure after birth. Fluorescence emission of the only tryptophan residue present in SP-B analogue (SP-C analogue has none) could in principle be exploited to probe SP-B analogue conformation, localization and interaction with other components of the pharmaceutical formulation. However, the high light scattering activity of the multi-lamellar vesicles suspension characterizing the pharmaceutical surfactant formulation represents a challenge for such studies. We show here that quenching of tryptophan fluorescence and Singular Value Decomposition analysis can be used to accurately calculate and subtract background scattering. The results indicate, with respect to Trp microenvironment, a conformationally homogeneous population of SP-B. Trp is highly accessible to the water phase, suggesting a surficial localization on the membrane of phospholipid vesicles, similarly to what observed for full length SP-B in natural lung surfactant, and supporting an analogous role in protein anchoring to the lipid phase

Fluorescence quantification of allantoin in biological samples by cap-immobilized allantoinase/resorcinol assay

Allantoin, the final product of urate degradation in non-hominoid mammals, forms in vivo through an enzyme-dependent pathway whose restoration has been proposed for the treatment of hyperuricemic conditions. Non-enzymatic urate oxidation to allantoin also occurs, making it a potential biomarker of oxidative stress in human physiology and pathology. This justifies the quest for fast and accurate methods for allantoin quantification overcoming the limitations of the current approaches, ranging from chromatographic to mass spectrometry techniques. We propose a simple fluorimetric assay that includes an enzymatic step for the conversion of S-allantoin to allantoate, followed by acid hydrolysis of the latter into glyoxylate in the presence of resorcinol. The lactone forming upon reaction of glyoxylate with resorcinol, in basic conditions, exhibits strong and stable fluorescence emission in the visible range. The assay proved to be specific for allantoin, with a linear response in the range of physiological values for several biological fluids, such as serum, urine, and saliva. We also verified the lack of reactivity by the most common interferents, including urate and its degradation intermediates. The assay, which does not require sophisticated instrumentation other than a conventional spectrofluorometer or a lab bench plate reader, was implemented on a solid support, exploiting enzyme encapsulation in a silica gel matrix, to allow reusability and improve stability over time

Regulation of human serine racemase activity and dynamics by halides, ATP and malonate

d-Serine is a non-proteinogenic amino acid that acts as a co-Agonist of the NMDA receptors in the central nervous system. d-Serine is produced by human serine racemase (hSR), a homodimeric pyridoxal 5?-phosphate (PLP)-dependent enzyme that also catalyzes the physiologically relevant β-elimination of both l-and d-serine to pyruvate and ammonia. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, we found that the catalytic activity is affected by halides, in the order fluoride < chloride < bromide. On the contrary, iodide elicited a complete inhibition, accompanied by a modulation of the tautomeric equilibrium of the internal aldimine. We also investigated the reciprocal effects of ATP and malonate, an inhibitor that reversibly binds at the active site, 20 A away from the ATP-binding site. ATP increased ninefold the affinity of hSR for malonate and malonate increased 100-fold that of ATP, confirming an allosteric interaction between the two binding sites. To further investigate this allosteric communication, we probed the active site accessibility by quenching of the coenzyme fluorescence in the absence and presence of ATP. We found that ATP stabilizes a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. METHODS: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. RESULTS: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. CONCLUSIONS: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia