Development of an easy-to-set-up multiple heart-cutting achiral-chiral LC-LC method for the analysis of branched-chain amino acids in commercial tablets.

In this paper, the development and application of a multiple heart-cutting achiral-chiral LC-LC method (mLC-LC) for the analysis of dansylated (Dns) branched-chain amino acids in commercial tablets are described. In the first dimension, a Waters Xbridge RP C18 achiral column was used under gradient conditions with buffered aqueous solution and acetonitrile. The elution order Dns-valine (Dns-Val) < Dns-isoleucine (Dns-Ile) < Dns-leucine (Dns-Leu) turned out with full resolution between adjacent peaks: 7.25 and 1.50 for the Val/Ile and the Ile/Leu pairs, respectively. A "research" validation study was performed, revealing high accuracy (Recovery%) and precision (RSD%) using two external set solutions, respectively, in the range 93.7%-104.1% and 0.4%-3.2%. The C18 column was connected via a two-position six-port switching valve to the quinidine-based Chiralpak quinidine-anion-exchange chiral column. A water/acetonitrile, 30/70 (v/v) with 50 mM ammonium acetate (apparent pH of 5.5) eluent allowed getting the three enantiomers' pairs resolved: RS equal to 4.3 for Dns-Val and Dns-Ile, and 1.7 for Dns-Leu. The application of the mLC-LC method confirmed that the content of Val, Ile, and Leu in the tablets was compliant with that labeled by the producer. Only l-enantiomers were found in the food supplement, as confirmed by LC-MS/MS analysis.

Discovery and Structure-Activity Relationships of Novel ssDAF-12 Receptor Modulators.

The nuclear receptor ssDAF-12 has been recognized as the key molecular player regulating the life cycle of the nematode parasite Strongyloides stercoralis. ssDAF-12 ligands permit the receptor to function as an on/off switch modulating infection, making it vulnerable to therapeutic intervention. In this study, we report the design and synthesis of a set of novel dafachronic acid derivatives, which were used to outline the first structure-activity relationship targeting the ssDAF-12 receptor and to unveil hidden properties shared by the molecular shape of steroidal ligands that are relevant to the receptor binding and modulation. Moreover, biological results led to the discovery of sulfonamide 3 as a submicromolar ssDAF-12 agonist endowed with a high receptor selectivity, no toxicity, and improved properties, as well as to the identification of unprecedented ssDAF-12 antagonists that can be exploited in the search for novel chemical tools and alternative therapeutic approaches for treating parasitism such as Strongyloidiasis.

Chemical exploration of TGR5 functional hot-spots: Synthesis and structure-activity relationships of C7- and C23-Substituted cholic acid derivatives.

The activation of TGR5 bestows on bile acids the ability to modulate nongenomic signaling pathways, which are responsible of physiological actions including immunosuppressive and anti-inflammatory properties as well as the regulation of glucose metabolism and energy homeostasis. TGR5 agonists have therefore emerged in drug discovery and preclinical appraisals as promising compounds for the treatment of liver diseases and metabolic syndrome. In this study, we have been devising site-selected chemical modifications of the bile acid scaffold to provide novel chemical tools able to modulate the functions of TGR5 in different tissues. Biological results of the tested collection of semisynthetic cholic acid derivatives were used to extend the structure-activity relationships of TGR5 agonists and to clarify the molecular basis and functional role of TGR5 hot-spots in the receptor activation and selectivity. Some unexpected properties deriving from the molecular structure of bile acids have been unveiled as relevant to the receptor activation and may hence be used to design novel, selective and potent TGR5 agonists.

Chiral high-performance liquid chromatography analysis of mono-, di-, and triacylglycerols with amylose- and cellulose-phenylcarbamate-based stationary phases.

The ever-increasing technological advancement in the (ultra)high-performance liquid chromatography tandem (high-resolution) mass spectrometry platforms have largely contributed to steeply intensify the interest towards lipidomics research. However, mass spectrometers alone are unable to distinguish between enantiomers. This obstacle is especially evident in the case of glycerolipids analysis due the prochiral nature of glycerol. Until a couple of decades ago, the stereoselective analysis of triacylglycerols (TAGs) was performed on the end products generated either from their enzymatic or chemical hydrolysis, namely on mono- or diacyl-sn-glycerols (MAGs and DAGs, respectively). These were then mostly analyzed with Pirkle-type chiral stationary phases (CSPs) after dedicated multi-step derivatization procedures. One of the most significant drawbacks of these traditional methods for enantioselective TAGs analysis (actually of the produced MAGs and DAGs, often investigated as target species per se) was the difficulty to totally abolish the migration of fatty acyls between glycerol positions. This made difficult to control and keep unaltered the stereochemistry of the original molecules. Over the last two decades, it has been widely demonstrated that the enantioselective analysis of intact TAGs as well as of non-derivatized MAGs and DAGs can be efficiently obtained using polysaccharide-based CSPs incorporating either amylose- or cellulose-phenylcarbamate derivatives chiral selectors. In this paper, the enantioselective methods developed with these CSPs for the enantioselective direct LC analysis of MAGs, DAGs and TAGs embedding different types of fatty acid residues are comprehensively reviewed.

Estimating the hydrophobicity extent of molecular fragments using reversed-phase liquid chromatography.

A fast HPLC method was developed to study the hydrophobicity extent of pharmaceutically relevant molecular fragments. By this strategy, the reduced amount of sample available for physico-chemical evaluations in early-phase drug discovery programs does not represent a limiting factor. The sixteen acid fragments investigated were previously synthesized also determining potentiometrically their experimental log D values. For four fragments it was not possible to determine such property since their values were outside of the instrumental working range (2 < pKa  < 12). An RP-HPLC method was therefore optimized. For each scrutinized method, some derived chromatographic indices were calculated, and Pearson's correlation coefficient (r) allowed to select the so-called "φ0 index" as the best correlating with the log D. The w s p H ${}_w/pH$ was fixed at 3.5 and a modification of some variables [organic modifier (methanol vs. ACN), stationary phase (octyl vs. octadecyl), presence/absence of the additives n-octanol, n-butylamine, and n-octylamine], allowed to select the best correlation conditions, producing a r = 0.94 (p < 0.001). Importantly, the φ0 index enabled the estimation of log D values for four fragments which were unattainable by potentiometric titration. Moreover, a series of molecular descriptors were calculated to identify the chemical characteristics of the fragments explaining the obtained φ0 . The number of hydrogen bond donors and the index of cohesive interaction correlated with the experimental data.

Turning a Tumor Microenvironment Pitfall into Opportunity: Discovery of Benzamidoxime as PD-L1 Ligand with pH-Dependent Potency.

PD-1/PD-L1 protein complex is attracting a great deal of interest as a drug target for the design of immune therapies able to block its assembly. Although some biologic drugs have entered clinical use, their poor response rate in patients are demanding further efforts to design small molecule inhibitors of PD-1/PD-L1 complex with higher efficacy and optimal physicochemical properties. Dysregulation of pH in the tumor microenvironment is indeed one of the key mechanisms promoting drug resistance and lack of response in cancer therapy. Integrating computational and biophysical approaches, herein we report a screening campaign that has led to identifying VIS310 as a novel ligand of PD-L1, with physicochemical properties enabling a pH-dependent binding potency. Additional optimization efforts by analogue-based screening have been instrumental to disclosing VIS1201, which exhibits improved binding potency against PD-L1 and is able to inhibit PD-1/PD-L1 complex formation in a ligand binding displacement assay. While providing preliminary structure-activity relationships (SARs) of a novel class of PD-L1 ligands, our results lay the foundation for the discovery of immunoregulatory small molecules resilient to tumor microenvironmental conditions for escaping drug-resistance mechanisms.

Microsampling and enantioselective liquid chromatography coupled to mass spectrometry for chiral bioanalysis of novel psychoactive substances.

In this paper, the development of efficient enantioselective HPLC methods for the analysis of five benzofuran-substituted phenethylamines, two substituted tryptamines, and three substituted cathinones is described. For the first time, reversed-phase (eluents made up with acidic water-methanol solutions) and polar-ionic (eluent made up with an acetonitrile-methanol solution incorporating both an acidic and a basic additive) conditions fully compatible with mass spectrometry (MS) detectors were applied with a chiral stationary phase (CSP) incorporating the (+)-(18-crown-6)-tetracarboxylic acid chiral selector. Enantioresolution was achieved for nine compounds with α and RS factors up to 1.32 and 5.12, respectively. Circular dichroism (CD) detection, CD spectroscopy in stopped-flow mode and quantum mechanical (QM) calculations were successfully employed to investigate the absolute stereochemistry of mephedrone, methylone and butylone and allowed to establish a (R)<(S) enantiomeric elution order for these compounds on the chosen CSP. Whole blood miniaturized samples collected by means of volumetric absorptive microsampling (VAMS) technology and fortified with the target analytes were extracted following an optimized protocol and effectively analysed by means of an ultra-high performance liquid chromatography-MS system. By this way a proof-of-concept procedure was applied, demonstrating the suitability of the method for quali-quantitative enantioselective assessment of the selected psychoactive substances in advanced biological microsamples. VAMS microsamplers including a polypropylene handle topped with a small tip of a polymeric porous material were used and allowed to volumetrically collect small aliquots of whole blood (10 µL) independently from its density. Highly appreciable volumetric accuracy (bias, in the -8.7-8.1% range) and precision (% CV, in the 2.8-5.9% range) turned out.

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements.

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral-chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.

Novel analgesic/anti-inflammatory agents: 1,5-Diarylpyrrole nitrooxyethyl sulfides and related compounds as Cyclooxygenase-2 inhibitors containing a nitric oxide donor moiety endowed with vasorelaxant properties.

The design of compounds able to combine the selective inhibition of cyclooxygenase-2 (COX-2) with the release of nitric oxide (NO) is a promising strategy to achieve potent anti-inflammatory agents endowed with an overall safer profile and reduced toxicity upon gastrointestinal and cardiovascular systems. With the aim of generating novel and selective COX-2 inhibiting NO-donors (CINOD) and encouraged by the promising results obtained with our nitrooxy- and hydroxyethyl ethers 11 and 12 reported in previous works, we shifted our attention on the synthesis of isosteric thioanalogs nitrooxy- and hydroxy ethyl sulfides 13a-c and 14a-c, respectively, along with their oxidation products nitrooxy- and hydroxyethyl sulfoxides 15a-c and 16a-c, respectively, also referred to as thio-CINOD. Preliminary data and metabolic analysis highlighted how the isosteric substitution of the ethereal oxygen atom of 11a-c with sulfur in compounds 13a-c, independently from the presence and the number of fluorine atoms in N1-phenyl ring, leads to new selective and highly potent COX-2 inhibitors, capable to induce vasorelaxant responses in vivo. The same behavior is observed with their oxidized counterparts nitrooxyethyl sulfoxides 15a-c, in which the oxidation state of the sulfur atom and the presence of the additional oxygen atom play a substantial role in enhancing compounds activity and vasorelaxation. In addition, the screened compounds proved significantly efficacious in mouse models of inflammation and nociception at the dose of 20 mg/kg.

Elucidation of retention mechanism of dipeptides on a ristocetin A-based chiral stationary phase using a combination of chromatographic and molecular simulation techniques.

Two chiral stationary phases virtually reproducing the Nautilus-R column were modeled in silico to study the enantiorecognition mechanism of some selected dipeptides, taking into consideration the two different anchoring alternatives to the silica layer involving the two ristocetin A amino groups. A mobile phase composed of water-methanol (40:60, v/v) was included in the system. The analyses of the trajectories supported the experimental L(LL)

Critical Assessment of a Structure-Based Screening Campaign for IDO1 Inhibitors: Tips and Pitfalls.

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.

Structural Basis of Human Dimeric α-Amino-ß-Carboxymuconate-ε-Semialdehyde Decarboxylase Inhibition With TES-1025.

Human α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) stands at a branch point of the de novo NAD+ synthesis pathway and plays an important role in maintaining NAD+ homeostasis. It has been recently identified as a novel therapeutic target for a wide range of diseases, including inflammatory, metabolic disorders, and aging. So far, in absence of potent and selective enzyme inhibitors, only a crystal structure of the complex of human dimeric ACMSD with pseudo-substrate dipicolinic acid has been resolved. In this study, we report the crystal structure of the complex of human dimeric ACMSD with TES-1025, the first nanomolar inhibitor of this target, which shows a binding conformation different from the previously published predicted binding mode obtained by docking experiments. The inhibitor has a K i value of 0.85 ± 0.22 nM and binds in the catalytic site, interacting with the Zn2+ metal ion and with residues belonging to both chains of the dimer. The results provide new structural information about the mechanism of inhibition exerted by a novel class of compounds on the ACMSD enzyme, a novel therapeutic target for liver and kidney diseases.

Efficient enantioresolution of aromatic α-hydroxy acids with Cinchona alkaloid-based zwitterionic stationary phases and volatile polar-ionic eluents.

Single enantiomers of mandelic acid (1), 3-phenyllactic acid (2), and 3-(4-hydroxyphenyl)lactic acid (3) are the subject of many fields of investigation, spanning from the pharmaceutical synthesis to that of biocompatible and biodegradable polymers, while passing from the interest towards their antimicrobial activity to their role as biomarkers of particular pathological conditions or occupational exposures to specific xenobiotics. All above mentioned issues justify the need for accurate analytical methods enabling the correct determination of the individual enantiomers. So far, all the developed liquid chromatography (LC) methods were not or hardly compatible with mass spectrometry (MS) detection. In this paper, a commercially available Cinchona-alkaloid derivative zwitterionic chiral stationary phase [that is, the CHIRALPAK® ZWIX(-)] was successfully used to optimize the enantioresolution of compounds 1-3 under polar-ionic (PI) conditions with a mobile phase consisting of an acetonitrile/methanol 95/5 (v/v) mixture with 80 mM formic acid. With the optimized conditions, enantioseparation and enantioresolution values up to 1.46 and 4.41, respectively, were obtained. In order to assess the applicability of the optimized enantioselective chromatography conditions in real-life scenarios and on MS-based systems, a proof-of-concept application was efficiently carried out by analysing dry urine spot samples spiked with 1 by means of a LC-MS system. The (S)<(R) enantiomer elution order (EEO) was established for compounds 1 and 2 by analysing a pure enantiomeric standard of known configuration. This was not possible for 3 because not commercially available. For this compound, the same EEO was identified applying a procedure based on ab initio time-dependent density-functional theory simulations coupled to electronic circular dichroism analyses. Moreover, a molecular dynamics simulation unveiled the role of the phenolic OH in compound 3 in the retention mechanism.

Recent advances in urea- and thiourea-containing compounds: focus on innovative approaches in medicinal chemistry and organic synthesis.

Urea and thiourea represent privileged structures in medicinal chemistry. Indeed, these moieties constitute a common framework of a variety of drugs and bioactive compounds endowed with a broad range of therapeutic and pharmacological properties. Herein, we provide an overview of the state-of-the-art of urea and thiourea-containing pharmaceuticals. We also review the diverse approaches pursued for (thio)urea bioisosteric replacements in medicinal chemistry applications. Finally, representative examples of recent advances in the synthesis of urea- and thiourea-based compounds by enabling chemical tools are discussed.

Synthesis and biological activity of cyclopropyl Δ7-dafachronic acids as DAF-12 receptor ligands.

The four cyclopropyl stereoisomers of Δ7-dafachronic acids were prepared from the bile acid hyodeoxycholic acid and employed as chemical tools to exploit the importance of the orientation and spatial disposition of the carboxyl tail and the C25-methyl group for the binding at the DAF-12 receptor. The synthesis route was based on (a) Walden inversion and stereoselective PtO2-hydrogenation to convert the L-shaped 5ß-cholanoid scaffold into the planar 5α-sterol intermediate; (b) two-carbon homologation of the side chain by Wittig and cyclopropanation reaction; and (c) formation of the 3-keto group and Δ7 double bond. The synthesized isomers were isolated and tested for their activity as DAF-12 ligands by AlphaScreen assays. Results showed a significant loss of potency and efficacy for all the four stereoisomers when compared to the parent endogenous ligand. Computational analysis has evidenced the configurational and conformational arrangement of both the carboxylic and the C25-methyl group of dafachronic acids as key structural determinants for DAF-12 binding and activation.

Original enantioseparation of illicit fentanyls with cellulose-based chiral stationary phases under polar-ionic conditions.

Fentanyl analogues used in therapy and a range of highly potent non-pharmaceutical fentanyl derivatives are subject to international control, as the latter are increasingly being synthesized illicitly and sold as 'synthetic heroin', or mixed with heroin. A significant number of hospitalizations and deaths have been reported in the EU and USA following the use of illicitly synthesized fentanyl derivatives. It has been unequivocally demonstrated that the enantiomers of fentanyl derivatives exhibit different pharmaco-toxicological profiles, which makes crucial to avail of suitable analytical methods enabling investigations at a "stereochemical level". Chromatographic methods useful to discriminate the enantioseparation of fentanyls and their derivatives are still missing in the literature. This is the first study in which the enantioseparation of four fentanyl derivatives, that is, (±)-trans-3-methyl norfentanyl, (±)-cis-3-methyl norfentanyl, ß-hydroxyfentanyl, and ß-hydroxythiofentanyl, has been obtained under polar-ionic conditions. Indeed, the use of ACN-based mobile phases with minor amounts of either 2-propanol or ethanol (plus diethylamine and formic acid as ionic additives) allowed obtaining enantioseparation and enantioresolution factors up to 1.83 and 7.02, respectively. For the study, the two chiral stationary phases cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate) were used, displaying a remarkably different performance towards the enantioseparation of (±)-cis-3-methyl norfentanyl. Chiral LC analyses with a high-resolution mass spectrometry detector were also carried out in order to confirm the obtained data and demonstrate the suitability and compatibility of the optimized mobile phases with mass spectrometric systems.

Fragment based drug design and diversity-oriented synthesis of carboxylic acid isosteres.

The medicinal chemist toolbox is plenty of (bio)isosteres when looking for a carboxylic acid replacement. However, systematic assessment of acid surrogates is often time consuming and expensive, while prediction of both physicochemical properties (logP and logD) as well as acidity would be desirable at early discovery stages for a better analog design. Herein in this work, to enable decision making on a project, we have synthesized by employing a Diversity-Oriented Synthetic (DOS) methodology, a small library of molecular fragments endowed with acidic properties. By combining in-silico and experimental methodologies these compounds were chemically characterized and, particularly, with the aim to know their physicochemical properties, the aqueous ionization constants (pKa), partition coefficients logD and logP of each fragment was firstly estimated by using molecular modeling studies and then validated by experimental determinations. A face to face comparison between data and the corresponding carboxylic acid might help medicinal chemists in finding the best replacement to be used. Finally, in the framework of Fragment Based Drug Design (FBDD) the small library of fragments obtained with our approach showed good versatility both in synthetic and physico-chemical properties.

Integrating experimental and computational techniques to study chromatographic enantioresolutions of chiral tetrahydroindazole derivatives.

With the selection of partially saturated 2H-indazoles as model compounds, we demonstrate the possibility to use Whelk-O1 chiral stationary phases (CSPs) to succeed in efficient small-scale preparative enantioseparations. Runs of three consecutive liquid chromatography injections (about 300 µg of racemate repeatedly injected in a 100 µL loop) produced groups of peaks without band contamination (α = 1.2 and RS = 2.57). With this procedure approximately 3.0 mg of each enantiomer, with enantiomeric excess ≥ 97% were obtained. Very profitably, the high volatility of n-hexane used as the sole eluent facilitated the solvent evaporation after the enantiomer recovery. High resolution mass spectrometry analysis confirmed that the chemical identity of the two enantiomers was preserved along the entire process. The ability of Whelk-O1 phases in enantioseparating structurally similar compounds was confirmed with the analysis of other two racemates. Moreover, the relevant chemoselectivity exhibited by the CSP towards the three racemates should allow to simultaneously optimizing the enantioselectivity of different analytes and perform small-scale enantioresolutions of different compounds during the same run. In this study, the integration of experimental off-line electronic circular dichroism analysis with ab initio time-dependent density-functional theory simulations facilitated the assignment of the absolute configuration of the single enantiomers, while a molecular dynamics protocol can be useful to make a priori predictions of the enantioseparation ability of CSP towards selected compounds.

Fragment-based Design of Zwitterionic, Strong Cation- and Weak Anion-Exchange Type Mixed-mode Liquid Chromatography Ligands and their Chromatographic Exploration.

The role of individual functional groups has been assessed with regard to surface charge and chromatographic retention. Coatings were prepared from various fragments of the chiral zwitterionic materials Chiralpak ZWIX(+) and ZWIX(-). The different chromatographic ligands allowed fine tuning of the surface charge. Chiralpak ZWIX phases showed strongly negative ζ-potentials over the entire pH-range. Zwitterionic congeners with quinuclidine and sulfonic acid moieties but lacking the quinolone ring in the ligand structure exhibited shifted ζ-potentials of around + 5 to 20 mV depending on the surrounding residues. Capillary electrophoretic mobilitiy measurements with the chromatographic ligands and molecular dynamics simulations were carried out to offer some explanation of these surface charge differences of the distinct zwitterionic stationary phases. The new mixed-mode phases were also chromatographically characterized by simple RP and HILIC tests. The results allowed their positioning within a large variety of different commercially available RP, HILIC and mixed-mode phases, which were evaluated as well, by multivariate data processing using principal component analysis. The new mixed-mode phases overall exhibit reasonable hydrophilicity-lipophilicity balance and enable retention of ionic compounds by additional ionic interactions through weak anion-exchange (WAX-type), strong cation-exchange (SCX-type) or both (RP/ZWIX-type). Hence, the new RP/ZWIX phases can be flexible tools for selectivity tuning in RP and HILIC separations.

Profiling calcium-dependent interactions between Sorcin and intrinsically disordered regions of human proteome.

BACKGROUND: Sorcin is a calcium sensor that exerts many calcium-related functions in the cells, e.g. it regulates calcium concentration in the cytoplasm, endoplasmic reticulum (ER) and mitochondria, by interacting with calcium pumps, exchangers and channels. Albeit Sorcin is an interesting potential cancer target, little is known about its interactors upon calcium-mediated activation. Our previous study suggested that Sorcin may recognize short linear binding motifs as the crystal structure revealed a self-interaction with a GYYPGG stretch in its N-terminus, and combinatorial peptide-phage display provided support for peptide-mediated interactions. METHODS: In this study we screened for motif-based interactions between Sorcin and intrinsically disordered regions of the human proteome using proteomic peptide phage display (ProP-PD). We identified a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a potential novel interactor and confirm the interaction through biophysical and cell-based approaches, and provide structural information through molecular dynamics simulations. RESULTS: Altogether, we identify a preferred motif in the enriched pool of binders and a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a preferred ligand. CONCLUSION: Through this study we gain information on a new Sorcin binding partner and profile Sorcin's motif-based interaction. GENERAL SIGNIFICANCE: The interaction between Sorcin and PPP1R3G may suggest a close dependence between glucose homeostasis and calcium concentration in the different cell compartments, opening a completely new and interesting scenery yet to be fully disclosed.