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Cancer neoantigens (nAgs) have the potential to elicit strong and tumor-specific immune responses and are, therefore, of great interest for cancer immunotherapeutic strategies, including therapeutic vaccines. Here we developed a novel neoantigen cancer vaccine based on adenoviruses derived from non-human Great Apes (GAds). GAds vaccination was highly effective in prophylactic or early therapeutic treatment of mouse tumors, independently of the number of encoded nAgs. In presence of high tumor burden, GAd has no antitumor effect unless combined with anti-PD1 treatment. In this more stringent setting, effectiveness of vaccination required the targeting of many neoantigens. Analysis of gene expression profile of tumors from responder mice showed greater diversification of the T cell repertoire with increased number of clonotypes in combo treated animals compared to anti-PD-1. Data suggest that GAd vaccines encoding a large number of nAgs can synergize with checkpoint inhibitors therapy by increasing the breadth of nAgs-specific T cells. Citation Format: Anna Morena D'Alise, Guido Leoni, Gabriella Cotugno, Fulvia Troise, Francesca Langone, Imma Fichera, Maria De Lucia, Rosa Vitale, Adriano Leuzzi, Veronica Bignone, Elena Di Matteo, Fabio Giovanni Tucci, Lidia Avalle, Valeria Poli, Armin Lahm, Maria Teresa Catanese, Antonella Folgori, Stefano Colloca, Alfredo Nicosia, Elisa Scarselli. A cancer vaccine targeting many neoantigens is required for effective eradication of large tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B105.

Fraxamoside, a macrocyclic secoiridoid glucoside featuring a hydroxytyrosol group, was recently identified as a xanthine oxidase inhibitor (XOI) comparable in potency in vitro to the standard antigout drug allopurinol. However, this activity and its considerably higher value than its derivatives oleuropein, oleoside 11-methyl ester, and hydroxytyrosol are not explained by structure–activity relationships (SARs) of known XOIs. To exclude allosteric mechanisms, we first determined the inhibition kinetic of fraxamoside. The resulting competitive mechanism prompted a computational SAR characterization, combining molecular docking and dynamics, which fully explained the behavior of fraxamoside and its derivatives, attributed the higher activity of the former to conformational properties of its macrocycle, and showed a substantial contribution of the glycosidic moiety to binding, in striking contrast with glycoside derivatives of most other XOIs. Overall, fraxamoside emerged as a lead compound for a new class of XOIs potentially characterized by reduced interference with purine metabolism.

Neuroglobin and cellular prion protein (PrPC) are expressed in the nervous system and co-localized in the retinal ganglion cell layer. Both proteins do not have an unambiguously assigned function, and it was recently reported that PrPC aggregates rapidly in the presence of neuroglobin, whereas it does not aggregate in the presence of myoglobin, another globin with different tissue specificity. Electrostatic complementarity between the unstructured PrPCN-terminus and neuroglobin has been proposed to mediate this specific interaction. To verifythis hypothesis experimentally, we have used a combined approach of automated docking and molecular dynamics (MD) studies carried out on short stretches of prion protein (PrP) N-terminus to identify the minimal electrostatically interacting aminoacidic sequences with neuroglobin. Subsequently, we have performed the synthesis of these peptides by solid phase methods, and we tested their interaction with neuroglobin by surface plasmon resonance (SPR). Preliminary results confirm unequivocally the specific interaction between synthetic PrP peptides and neuroglobin suggesting a crucial role of PrPC positively charged regions in thisprotein–protein association. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Irreversible aggregation limits bioavailability and therapeutic activity of protein-based drugs. Here we show that an aggregation- resistant mutant can be engineered by structural homology with a non-amyloidogenic analogue and that the aggregation-resistant variant may act as an inhibitor. This strategy has successfully been applied to the amyloidogenic human calcitonin (hCT). Including only five residues from the non-amyloidogenic salmon calcitonin (sCT), we obtained a variant, polar human calcitonin (phCT), whose solution structure was shown by CD, NMR, and calculations to be practically identical to that of sCT. phCT was also observed to be a potent amyloidogenesis inhibitor of hCT when mixed with it in a 1:1 ratio. Fibrillation studies of phCT and the phCT-hCT mixture mimicked the sCT behavior in the kinetics and shapes of the fibrils with a dramatic reduction with respect to hCT. Finally, the effect of phCT alone and of the mixture on the intracellular cAMP level in T47D cells confirmed for the mutant and the mixture their calcitonin-like activity, exhibiting stimulation effects identical to those of sCT, the current therapeutic form. The strategy followed appears to be suitable to develop new forms of hCT with a striking reduction of aggregation and improved activity. Finally, the inhibitory properties of the aggregation- resistant analogue, if confirmed for other amyloidogenic peptides, may favor a new strategy for controlling fibril formation in a variety of human diseases.

In this work the thermophilic trehalose/maltose-binding protein from Thermococcus litoralis is presented as a probe for the design of a high stable fluorescence biosensor for glucose. In particular, we show the possibility of modulating the protein specificity by changing temperature. In addition to glucose sensing, we also report on the possibility of utilizing odorant-binding proteins as a probe for the development of optical sensors for analytes of environmental interests.

A tri-functional monomeric primase–polymerase domain encoded by the plasmid pIT3 from Sulfolobus solfataricus strain IT3 was identified using a structural–functional approach. The N-terminal domain of the pIT3 replication protein encompassing residues 31–245 (i.e. Rep245) was modeled onto the crystallographic structure of the bifunctional primase–polymerase domain of the archaeal plasmid pRN1 and refined by molecular dynamics in solution. The Rep245 protein was purified following overexpression in Escherichia coli and its nucleic acid synthesis activity was characterized. The biochemical properties of the polymerase activity such as pH, temperature optima and divalent cation metal dependence were described. Rep245 was capable of utilizing both ribonucleotides and deoxyribonucleotides for de novo primer synthesis and it synthesized DNA products up to several kb in length in a template-dependent manner. Interestingly, the Rep245 primase–polymerase domain harbors also a terminal nucleotidyl transferase activity, being able to elongate the 3′-end of synthetic oligonucleotides in a non-templated manner. Comparative sequence–structural analysis of the modeled Rep245 domain with other archaeal primase–polymerases revealed some distinctive features that could account for the multifaceted activities exhibited by this domain. To the best of our knowledge, Rep245 typifies the shortest functional domain from a crenarchaeal plasmid endowed with DNA and RNA synthesis and terminal transferase activity.

2-Substituted-5-nitro-benzenesulfonamides incorporating a large variety of secondary/tertiary amines were explored as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), with the aim of designing bioreductive inhibitors targeting the hypoxia overexpressed, tumor-associated isozymes. The compounds were ineffective inhibitors of the cytosolic isoform I, showed a better inhibition of the physiologically relevant CA II (KIs of 8.8-4975 nM), and strongly inhibited the tumor-associated CA IX and XII (KIs of 5.4-653 nM). Some of these compounds showed excellent selectivity ratios for the inhibition of the tumor-associated isozymes over the cytosolic ones (in the range of 10-1395). The X-ray crystal structure of the adduct of hCA II with the lead molecule 2-chloro-5-nitro-benzenesulfonamide as well as molecular modeling studies for interaction with hCA IX afforded a better understanding of factors governing the discrimination of the two isoforms for this type of bioreductive compound targeting specifically hypoxic tumors.

Avarol is a marine sesquiterpenoid hydroquinone, previously isolated from the marine sponge Dysidea avara Schmidt (Dictyoceratida), with antiinflammatory, antitumor, antioxidant, antiplatelet, anti-HIV, and antipsoriatic effects. Recent findings indicate that some thio-avarol derivatives exhibit acetylcholinesterase (AChE) inhibitory activity. The multiple pharmacological properties of avarol, thio-avarol and/or their derivatives prompted us to continue the in vitro screening, focusing on their AChE inhibitory and neuroprotective effects. Due to the complex nature of Alzheimer's disease (AD), there is a renewed search for new, non hepatotoxic anticholinesterasic compounds. This paper describes the synthesis and in vitro biological evaluation of avarol-3′-thiosalicylate (TAVA) and thiosalycil-prenyl-hydroquinones (TPHs), as non hepatotoxic anticholinesterasic agents, showing a good neuroprotective effect on the decreased viability of SHSY5Y human neuroblastoma cells induced by oligomycin A/rotenone and okadaic acid. A molecular modeling study was also undertaken on the most promising molecules within the series to elucidate their AChE binding modes and in particular the role played by the carboxylate group in enzyme inhibition. Among them, TPH4, bearing a geranylgeraniol substituent, is the most significant Electrophorus electricus AChE (EeAChE) inhibitor (IC = 6.77 ± 0.24 μM), also endowed with a moderate serum horse butyrylcholinesterase (eqBuChE) inhibitory activity, being also the least hepatotoxic and the best neuroprotective compound of the series. Thus, TPHs represents a new family of synthetic compounds, chemically related to the natural compound avarol, which has been discovered for the potential treatment of AD. Findings prove the relevance of TPHs as a new possible generation of competitive AChE inhibitors pointing out the importance of the salycilic substituents on the hydroquinone ring. Since these compounds do not belong to the class of alkaloids, which are notorious for their capability to inhibit AChE while exhibiting side effects, they may constitute novel active AChE inhibitors with fewer side effects., MJOG thanks MINECO (Spain) for grant SAF2010-20337, and UCM for grant GR3/14. JMC thanks MINECO (Spain) (SAF2012- 33304 and SAF2015-56685-R). for continued support. This work was partially supported by the International Program “Short Term Mobility (S.T.M.)” of CNReNational Research Council of Italy.

[object Object]Tetrahydroisoquinoline derivatives containing embedded urea functions were identified as selective TRPM8 channel receptor antagonists. Structure-activity relationships were investigated, with the following conclusions: (a) The urea function and the tetrahydroisoquinoline system are necessary for activity. (b) Bis(1-aryl-6,7dimethoxy-1,2,3,4- tetrahydroisoquinolyl)ureas are more active than compounds containing one tetrahydroisoquinoline ring and than an open phenetylamine ureide. (c) Trans compounds are more active than their cis isomers. (d) Aryl substituents are better than alkyls at the isoquinoline C-1 position. (e) Electron-withdrawing substituents lead to higher activities. The most potent compound is the 4-F derivative, with IC50 in the 10-8 M range and selectivities around 1000:1 for most other TRP receptors. Selected compounds were found to be active in reducing the growth of LNCaP prostate cancer cells. TRPM8 inhibition reduces proliferation in the tumor cells tested but not in nontumor prostate cells, suggesting that the activity against prostate cancer is linked to TRPM8 inhibition.

The glycoterpenoid syphonoside (1) is the main secondary metabolite of both the marine mollusk Syphonota geographica and the sea-grass Halophila stipulacea, two Indo-Pacific species migrated to the Mediterranean Sea through the Suez Canal. The structure and the absolute stereochemistry of 1, which displays unique structural features, has been accomplished by using a combination of spectroscopic techniques, degradation reactions, and conformational analysis methods. Compound 1 was able to inhibit high density induced apoptosis in a number of human and murine carcinoma cell lines.

Zonisamide and topiramate are two antiepileptic drugs known to induce weight loss in epilepsy patients. These molecules were recently shown to act as carbonic anhydrase (CA) inhibitors, being presumed that the weight loss may be due to the inhibition of the mitochondrial isozymes CA VA and CA VB involved in metabolic processes, among which lipid biosynthesis. To better understand the interaction of these compounds with CAs, here, we report a homology modeling and molecular dynamics simulations study on their adducts with human carbonic anhydrase VA (hCA VA). According to our results, in both cases the inhibitor sulfamate/sulfonamide moiety participates in the canonical interactions with the catalytic zinc ion, whereas the organic scaffold establishes a large number of van der Waals and polar interactions with the active site cleft. A structural comparison of these complexes with the corresponding homologues with human carbonic anhydrase II (hCA II) provides a rationale to the different affinities measured for these drugs toward hCA VA and hCA II. In particular, our data suggest that a narrower active site cleft, together with a different hydrogen bond network arrangement of hCA VA compared to hCA II, may account for the different Kd values of zonisamide and topiramate toward these physiologically relevant hCA isoforms. These results provide useful insights for future design of more isozyme-selective hCA inhibitors with potential use as anti-obesity drugs possessing a novel mechanism of action.

Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges.

The validation of biomolecular computational approaches represents a critical step to assess their general reliability and possible fields of application. However, it requires a combined selection of protocols and representative model systems which needs great attention, since both choices can bias the results, or alter the method sensitivity to the numerous parameters that describe the systems. Here we propose the use of self-included monofunctionalized \(\beta \)-cyclodextrins (mf-\(\beta \)-CDs) to evaluate and compare solvation methods. For that end, we characterized the self-inclusion processes of three mf-\(\beta \)-CDs by molecular dynamics simulations performed with both explicit solvent and three implicit solvent implementations based on the generalized Born approach. In particular, we monitored the sensitivity to the different solvation models of the resulting trajectories and of several structural and energetic parameters usually adopted to study cyclodextrins or solvation methods. Our results confirmed that mf-\(\beta \)-CD are useful testbeds to compare solvation approaches because their properties are particularly affected by the relative stabilities of hydrophobic versus polar interactions and by energy contributions implicitly or explicitly dependent on exposed molecular surfaces.

We describe the X-ray diffraction structure of the terminally protected dipeptide amide Boc-Aib-L-Mag-NHBzl (Aib is alpha-aminoisobutyric acid and Mag is C-alpha-methyl, C-a-allyl-glycine) and the results of conformational energy computations on Ac-L-Mag-NHMe, Ac-Aib-L-Mag-NHMe, and Ac-L-Mag-Aib-NHMe. On the basis of these data, we performed conformational energy computations on the sequence -Aib-Xxx-(Aib)(2)-Xxxx-Aib- (Xxx = L-Mag) to check the feasibility of ring-closing metathesis, a currently extensively investigated reaction useful to enhance peptide helicity and metabolic stability, on this 3(10)-helix forming model hexamer with the two olefinic amino acids at the i, i+3 relative positions. Computations were extended to peptides based on olefinic residues with the side chains elongated by one (L-hMag) or two (L-hhMag) carbon atoms. A comparison was also made with peptides characterized by the related, non-C-alpha-methylated (C-alpha-trisubstituted) L-Agl (C-alpha-allyl-L-glycine), L-hAgl, and L-hhAgl residues. We conclude that, to achieve ring-closing metathesis with an unperturbed 3(10)-helical conformation and a symmetrical all-hydrocarbon tether, the side-chain length for each of the two i, i+3 olefinic amino acids requires at least five carbon atoms (hhMag or hhAgl), thereby producing an 18-atom macrocycle.

UV-visible absorption spectroelectrochemistry elucidated the different redox behaviours of Fe(III)- and Co(III)-mimochrome VI artificial enzymes, adsorbed on mesoporous conductive films of ITO. The reduction of the ferric complex was rapid and reversible, while the cobaltic complex exhibited irreversible processes probably related to multiple coordination states.

A series of 5-(carbamoylmethoxy)benzylidene-2-oxo/thioxo-4-thiazolidinone derivatives (6–9) were synthesized as inhibitors of aldose reductase (AR), enzyme which plays a crucial role in the development of diabetes complications as well as in the inflammatory processes associated both to diabetes mellitus and to other pathologies. In vitro inhibitory activity indicated that compounds 6–9a–d were generally good AR inhibitors. Acetic acid derivatives 8a–d and 9a–d were shown to be the best enzyme inhibitors among the tested compounds endowed with significant inhibitory ability levels reaching submicromolar IC50 values. Moreover, some representative AR inhibitors (7a, 7c, 9a, 9c, 9d) were assayed in cultures of human keratinocytes in order to evaluate their capability to reduce NF-kB activation and iNOS expression. Compound 9c proved to be the best derivative endowed with both interesting AR inhibitory effectiveness and ability to reduce NF-kB activation and iNOS expression. Molecular docking and molecular dynamics simulations were undertaken to investigate the binding modes of selected compounds into the active site of AR in order to rationalize the inhibitory effectiveness of these derivatives.

Here, we present a minimal hybrid ligand/receptor-based pharmacophore model (PM) for CXCR4, a chemokine receptor deeply involved in several pathologies, such as HIV infection, rheumatoid arthritis, cancer development/progression, and metastasization. This model, considerably simpler than those thus far proposed for this receptor, has been used to search for new CXCR4 inhibitors in a small marine natural product library available at ICB-CNR Institute (Pozzuoli, NA, Italy), since natural products, with their naturally selected chemical and functional diversity, represent a rich source of bioactive scaffolds; computational approaches allow searching for new scaffolds with a minimal waste of possibly precious natural product samples; and our "stripped-down" model substantially increases the probabilities of identifying potential hits even in small-sized libraries. This search, also validated by a systematic virtual screening of the same library, has led to the identification of a new CXCR4 ligand, phidianidine A (PHIA). Docking studies supported PHIA activity and suggested its possible binding modes to CXCR4. Using the CXCR4-expressing/CXCR7-negative GH4C1 cell line we show that PHIA inhibits CXCL12-induced DNA synthesis, cell migration, and ERK1/2 activation. The specificity of these effects was confirmed by the lack of PHIA activity in GH4C1 cells, in which siRNA highly reduces CXCR4 expression and the lack of cytoxicity of PHIA was also verified. Thus, PHIA represents a promising lead for a new family of CXCR4 modulators with wide margins of improvement in potency and specificity offered by the small and very simple underlying PM.

Mass Spectrometry (MS)-based strategies featuring chemical or biochemical probing represent powerful and versatile tools for studying structural and dynamic features of proteins and their complexes. In fact, they can be used both as an alternative for systems intractable by other established high-resolution techniques, and as a complementary approach to these latter, providing different information on poorly characterized or very critical regions of the systems under investigation (Russell et al., 2004). The versatility of these MS-based methods depends on the wide range of usable probing techniques and reagents, which makes them suitable for virtually any class of biomolecules and complexes (Aebersold et al., 2003). Furthermore, versatility is still increased by the possibility of operating at very different levels of accuracy, ranging from qualitative high-throughput fold recognition or complex identification (Young et al., 2000), to the fine detail of structural rearrangements in biomolecules after environmental changes, point mutations or complex formations (Nikolova et al.,1998; Millevoi et al., 2001; Zheng et al., 2007). However, these techniques heavily rely upon the availability of powerful computational approaches to achieve a full exploitation of the information content associated with the experimental data. The determination of three-dimensional (3D) structures or models by MS-based techniques (MS3D) involves four main activity areas: 1) preparation of the sample and its derivatives labelled with chemical probes; 2) generation of derivatives/fragments of these molecules for further MS analysis; 3) interpretation of MS data to identify those residues that have reacted with probes; 4) derivation of 3D structures consistent with information from previous steps. Ideally, this procedure should be considered the core of an iterative process, where the final model possibly prompts for new validating experiments or helps the assignment of ambiguous information from the mass spectra interpretation step. Both the overall MS3D procedure and its different steps have been the subject of several accurate review and perspective articles (Sinz, 2006; Back et al., 2003; Young et al., 2000; Friedhoff, 2005, Renzone, et al., 2007a). However, with the partial exception of a few recent papers (Van Dijk et al., 2005; Fabris et al., 2010; Leitner et al., 2010), the full computational detail behind 3D model building (step 4) has generally received less attention than the former three steps. Structural derivation in MS3D, in fact, is considered a special case of structural determination from sparse/indirect constraints (SD-SIC). Nevertheless, information for modelling derivable from MS-based experiments exhibits some peculiar

Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity. Herein, we describe the spectroscopic and functional characterization of Fe(III)-mimochrome VI, a 3.5 kDa synthetic heme-protein model, which displays a peroxidase-like catalytic activity. By the use of hydrogen peroxide, Fe(III)-mimochrome VI efficiently catalyzes the oxidation of several substrates, with a typical Michaelis-Menten mechanism and with several multiple turnovers. The catalytic efficiency of Fe(III)-mimochrome VI in the oxidation of 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and guaiacol (k(cat)/K(m)=4417 and 870 mM(-1) s(-1), respectively) is comparable to that of native horseradish peroxidase (HRP, k(cat)/K(m)=5125 and 500 mM(-1) s(-1), respectively). Fe(III)-mimochrome VI also converts phenol to 4- and 2-nitrophenol in the presence of NO(2) (-) and H(2) O(2) in high yields. These results demonstrate that small synthetic peptides can impart high enzyme activities to metal cofactors, and anticipate the possibility of constructing new biocatalysts tailored to specific functions.

A series of nine casbane diterpenes, compounds 5-13, exhibiting either cis or trans ring junctions were isolated from the Hainan soft coral Sinularia depressa. The structures of this group of compounds, the basic member of which was named depressin (5), were established by detailed spectroscopic analysis. In addition, the absolute configuration of the main metabolite, 10-hydroxydepressin (7), and of its epimer, 1-epi-10-hydroxydepressin (8), was determined by a combination of conformational analysis and the modified Mosher's method. A stereochemical relationship between all isolated molecules was investigated by analyzing their circular dichroism profiles. Antiproliferative and antibacterial activities of the depressins were also evaluated.

The multiple antibiotic resistance regulator (MarR) family constitutes a significant class of transcriptional regulators whose members control a variety of important biological functions such as regulation of response to environmental stress, control of virulence factor production, resistance to antimicrobial agents, and regulation of aromatic catabolic pathways. Although the majority of MarR family members have been characterized as transcriptional repressors, a few examples of transcriptional activators have also been reported. BldR is a newly identified member of this family that has been demonstrated to act as a transcriptional activator in stress response to aromatic compounds in the crenarchaeon Sulfolobus solfataricus. In this work, we report findings on the BldR X-ray crystal structure and present a molecular modeling study on the complex that this protein forms with its cognate DNA sequence, thus providing the first detailed description of the DNA-binding mechanism of an archaeal activator belonging to the MarR family. Two residues responsible for the high binding specificity of this transcriptional regulator were also identified. Our studies demonstrated that, in Archaea, the capability of MarR family members to act as activators or repressors is not related to a particular DNA-binding mechanism but rather could be due to the position of the binding site on the target DNA. Moreover, since genes encoding MarR proteins often control transcription of operons that encode for multisubstrate efflux pumps, our results also provided important insights for the identification of new tools to overcome the microorganism's multidrug resistance.

Two approaches were used to design inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1): the tail and the ring approaches. Aliphatic sulfamates constitute a class ofCAinhibitors (CAIs) that cannot be classified in either one of these categories. Wereport here the detailed inhibition profile of four such compounds against isoforms CAs I-XIV, the first crystallographic structures of these compounds in adduct with isoform II, and molecular modeling studies for their interaction with hCA IX. Aliphatic monosulfamates/bis-sulfamates were nanomolar inhibitors of hCAs II, IX, and XII, unlike aromatic/heterocyclic sulfonamides that promiscuously inhibit most CA isozymes with low nanomolar affinity. The bis-sulfamates incorporating 8 or 10 carbon atoms showed higher affinity for the tumor-associated hCA IX compared to hCA II, whereas the opposite was true for the monosulfamates. The explanation for their interaction with CA active site furnishes insights for obtaining compounds with increased affinity/selectivity for various isozymes. Introduction A paradigm in carbonic anhydrase (CA,a EC 4.2.1.1) drug design was that aliphatic sulfonamidesRSO2NH2 (R=aliphatic group) are inactive as inhibitors, unlike the aromatic/heterocyclic ones of the type ArSO2NH2.1 This view was subsequently challenged, being shown that aliphatic sulfonamides incorporating perhaloalkyl moieties of the type CnX2nþ1SO2- NH2 (n=1-4, X=F, Cl),2 as well as various aliphatic sulfamates/bis-sulfamates3-5 potently inhibit several CA isozymes involved in fundamental physiologic/pathologic states. Indeed, this family of metalloenzymes comprises 16 different isoforms, of which several are cytosolic (CAs I-III, CA VII, and CAXIII), five are membrane-bound (CA IV, CAIX, CA XII, CA XIV, and CA XV), two are mitochondrial (CAs VA and VB), and one (CA VI) is secreted into saliva/milk.6-13 Three acatalytic forms are also known, i.e., CA VIII, CA X, and CA XI.9 These enzymes are involved in crucial physiological processes connected with respiration and transport of CO2/bicarbonate betweenmetabolizing tissues and lungs, pH and CO2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions (such as gluconeogenesis, lipogenesis, and ureagenesis), bone resorption, calcification, tumorigenicity, and

The discovery of pharmaceutical agents is a complex, lengthy and costly process, critically depending on the availability of rapid and efficient screening methods. In particular, when targets are large, multidomain proteins, their complexity may affect unfavorably technical feasibility, costs and unambiguity of binding test interpretation. A possible strategy to overcome these problems relies on molecular design of receptor fragments that are: sensible targets for ligand screenings, conformationally stable also as standalone domains, easily synthesized and immobilized on chip for Biacore experiments. An additional desirable feature for new ligands is the ability of selectively targeting alternative conformational states typical of many proteins. To test the feasibility of such approach on a case with potential applicative interest, we developed a surface plasmon resonance (SPR)-based screening method for drug candidates toward HER2, a Tyr-kinase receptor targeted in anticancer therapies. HER2 was mimicked by HER2-DIVMP, a modified fragment of it immobilized onto the sensor surface specifically modeling HER2 domain IV in its bounded form, designed by structural comparison of HER2 alone and in complex with Herceptin, a monoclonal therapeutic anti-HER2 antibody. This design and its implementation in SPR devices was validated by investigating Herceptin- HER2-DIVMP affinity, measuring its dissociation constant (K(D)=19.2 nM). An efficient synthetic procedure to prepare the HER2-DIVMP peptide was also developed. The HER2-DIVMP conformational stability suggested by experimental and computational results, makes it also a valuable candidate as a mold to design new molecules selectively targeting domain IV of HER2.

Laura Tornatore, Simona M. Monti1, Daniela Marasco, Nina Dathan, Rosa M. Vitale, Ettore Benedetti, Carlo Pedone, Salvatore Papa, Guido Franzoso and Menotti Ruvo Istituto di Biostrutture e Bioimmagini CNR, via Mezzocannone, 16, Napoli, Italy; Universita Federico II di Napoli, via Mezzocannone, 16, 80134, Napoli, Italy; The Ben May Institute for Cancer Research and the University of Chicago, 24 East 57 Street, Chicago, IL60637.

The antimicrobial peptide distinctin consists of two peptide chains linked by a disulfide bridge; it presents a peculiar fold in water resulting from noncovalent dimerization of two heterodimeric molecules. To investigate the contribution of each peptide chain and the S-S bond to distinctin biochemical properties, different monomeric and homodimeric peptide analogues were synthesized and comparatively evaluated with respect to the native molecule. Our experiments demonstrate that the simultaneous occurrence of both peptide chains and the disulfide bond is essential for the formation of the quaternary structure of distinctin in aqueous media, able to resist protease action. In contrast, distinctin and monomeric and homodimeric analogues exhibited comparable antimicrobial activities, suggesting only a partial contribution of the S-S bond to peptide killing effectiveness. Relative bactericidal properties paralleled liposome permeabilization results, definitively demonstrating that microbial membranes are the main target of distinctin activity. Various biophysical experiments performed in membrane-mimicking media, before and after peptide addition, provided information about peptide secondary structure, lipid bilayer organization, and lipid-peptide orientation with respect to membrane surface. These data were instrumental in the generation of putative models of peptide-lipid supramolecular pore complexes.

Gadd45 alpha, beta, and gamma proteins, also known as growth arrest and DNA damage-inducible factors, have a number of cellular functions, including cell-cycle regulation and propagation of signals produced by a variety of cellular stimuli, maintaining genomic stability and apoptosis. Furthermore, Gadd45 beta has been indicated as a major player in the endogenous NF-kappa B-mediated resistance to apoptosis in a variety of cell lines. In fibroblasts this mechanism involves the inactivation of MKK7, the upstream activator of JNK, by direct binding within the kinase ATP pocket. On the basis of a number of experimental data, the structures of Gadd45 beta and the Gadd45 beta-MKK7 complex have been predicted recently and data show that interactions are mediated by acidic loops 1 and 2, and helices 3 and 4 of Gadd45 beta. Here, we provide further evidence that Gadd45 beta is a prevailingly alpha-helical protein and that in solution it is able to form non covalent dimers but not higher-order oligomers, in contrast to what has been reported for the homologous Gadd45 alpha. We show that the contact region between the two monomers is comprised of the predicted helix 1 (residues Q17-Q33) and helix 5 (residues K131-R146) of the protein, which appear to be antiparallel and to form a large dimerisation surface not involved in MKK7 recognition. The results suggest the occurrence of a large complex containing at least an MKK7-Gadd45 beta:Gadd45 beta -MKK7 tetrameric unit whose complexity could be further increased by the dimeric nature of the isolated MKK7

NF-kappaB/Rel factors control programmed cell death (PCD), and this control is crucial to oncogenesis, cancer chemoresistance, and antagonism of tumor necrosis factor (TNF) alpha-induced killing. With TNFalpha, NF-kappaB-mediated protection involves suppression of the c-Jun-N-terminal kinase (JNK) cascade, and we have identified Gadd45beta, a member of the Gadd45 family, as a pivotal effector of this activity of NF-kappaB. Inhibition of TNFalpha-induced JNK signaling by Gadd45beta depends on direct targeting of the JNK kinase, MKK7/JNKK2. The mechanism by which Gadd45beta blunts MKK7, however, is unknown. Here we show that Gadd45beta is a structured protein with a predicted four-stranded beta-sheet core, five alpha-helices, and two acidic loops. Association of Gadd45beta with MKK7 involves a network of interactions mediated by its putative helices alpha3 and alpha4 and loops 1 and 2. Whereas alpha3 appears to primarily mediate docking to MKK7, loop 1 and alpha4-loop 2 seemingly afford kinase inactivation by engaging the ATP-binding site and causing conformational changes that impede catalytic function. These data provide a basis for Gadd45beta-mediated blockade of MKK7, and ultimately, TNFalpha-induced PCD. They also have important implications for treatment of widespread diseases.

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Sso7d from the extreme thermophilic crenarchaeon Sulfolobus solfataricus is a multifunctional protein in in vitro assays, whose in vivo role is still puzzling. Crystals of Sso7d in complex with DNA elucidated the protein surface involved in the binding to the nucleic acid, whereas the locations of the Sso7d regions responsible for a chaperone activity in renaturing protein aggregates (i.e., the protein-binding surface and the site of ATPase activity) are still unknown. We identified the regions of Sso7d involved in protein-binding by limited proteolysis experiments associated to advanced mass spectrometric procedures performed on isolated Sso7d and Sso7d in complex with the peptide melittin. By affinity labeling of Sso7d with the ATP analogue 5′-p-fluorosulfonylbenzoyl adenosine and characterization of the labeled tryptic peptides by tandem mass spectrometry, we found that Y7 and K39 are residues involved in ATP binding/hydrolysis. Insights into the positions of the ligands melittin and ATP were achieved by a molecular modeling study; the models obtained were in agreement with most experimental data. A comparison among the complexes of Sso7d with DNA, with melittin, and with ATP showed that the DNA-binding surface and the protein-binding surface overlap, whereas the ATPase site is mostly independent of the binding sites for the nucleic acid and melittin. Proteins 2007. © 2007 Wiley-Liss, Inc.

The X-ray crystal structure of the fluorescent antitumor sulfonamide carbonic anhydrase (CA, EC, 4.2.1.1) inhibitor (4-sulfamoylphenylethyl)thioureido fluorescein (1) in complex with the cytosolic isoform hCA II is reported, together with a modeling study of the adduct of 1 with the tumor-associated isoform hCA IX. Its binding to hCA II is similar to that of other benzesulfonamides, with the ionized sulfonamide coordinated to the Zn2+ ion within the enzyme active site, and also participating in a network of hydrogen bonds with residues Thr199 and Glu106. The scaffold of 1 did not establish polar interactions within the enzyme active site but made hydrophobic contacts (

An approach for designing bioreductive, hypoxia-activatable carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the tumor-associated isoforms is reported. Sulfonamides incorporating 3,3'-dithiodipropionamide/2,2'-dithiodibenzamido moieties were prepared and reduced enzymatically/chemically in conditions present in hypoxic tumors, leading to thiols. The X-ray crystal structure of the most promising compound, 4-(2-mercaptophenylcarboxamido)benzenesulfonamide, which as disulfide showed a K(I) against hCA IX of 653 nM (in reduced form of 9.1 nM), in adduct with hCA II showed the inhibitor making favorable interactions with Gln92, Val121, Phe131, Leu198, Thr199, Thr200, Pro201, and Pro202, whereas the sulfamoyl moiety was coordinated to the Zn2+ ion. The same interactions were preserved in the adduct with hCA IX, but in addition, a hydrogen bond between the SH moiety of the inhibitor and the amide nitrogen of Gln67 was evidenced, which may explain the almost 2 times more effective inhibition of the tumor-associated isozyme over the cytosolic isoform.

Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure.

Cationic lipodepsipeptides from Pseudomonas spp. have been characterized for their structural and antimicrobial properties. In the present study, the structure of a novel lipodepsipeptide, cormycin A, produced in culture by the tomato pathogen Pseudomonas corrugata was elucidated by combined protein chemistry, mass spectrometry and two-dimensional NMR procedures. Its peptide moiety corresponds to L-Ser-D-Orn-L-Asn-D-Hse-L-His-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl) [where Orn represents ornithine, Hse is homoserine, aThr is allo-threonine, Z-Dhb is 2,3-dehydro-2-aminobutanoic acid, Asp(3-OH) is 3-hydroxyaspartic acid and Thr(4-Cl) is 4-chlorothreonine], with the terminal carboxy group closing a macrocyclic ring with the hydroxy group of the N-terminal serine residue. This is, in turn, N-acylated by 3,4-dihydroxy-esadecanoate. In aqueous solution, cormycin A showed a rather compact structure, being derived from an inward orientation of some amino acid side chains and from the ‘hairpin-bent’ conformation of the lipid, due to inter-residue interactions involving its terminal part. Cormycin was significantly more active than the other lipodepsipeptides from Pseudomonas spp., as demonstrated by phytotoxicity and antibiosis assays, as well as by red-blood-cell lysis. Differences in biological activity were putatively ascribed to its weak positive net charge at neutral pH. Planar lipid membrane experiments showed step-like current transitions, suggesting that cormycin is able to form pores. This ability was strongly influenced by the phospholipid composition of the membrane and, in particular, by the presence of sterols. All of these findings suggest that cormycin derivatives could find promising applications, either as antifungal compounds for topical use or as post-harvest biocontrol agents.

Cationic lipodepsipeptides from Pseudomonas spp. have been characterized for their structural and antimicrobial properties. In the present study, the structure of a novel lipodepsipeptide, cormycin A, produced in culture by the tomato pathogen Pseudomonas corrugata was elucidated by combined protein chemistry, mass spectrometry and two-dimensional NMR procedures. Its peptide moiety corresponds to L-Ser-D-Orn-L-Asn-D-Hse-L-His- L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl) [where Orn represents ornithine, Hse is homoserine, aThr is allo-threonine, Z-Dhb is 2,3- dehydro-2-aminobutanoic acid, Asp(3-OH) is 3-hydroxyaspartic acid and Thr(4-Cl) is 4-chlorothreonine], with the terminal carboxy group closing a macrocyclic ring with the hydroxy group of the N-terminal serine residue. This is, in turn, N-acylated by 3,4-dihydroxy-esadecanoate. In aqueous solution, cormycin A showed a rather compact structure, being derived from an inward orientation of some amino acid side chains and from the 'hairpinbent' conformation of the lipid, due to inter-residue interactions involving its terminal part. Cormycin was significantly more active than the other lipodepsipeptides from Pseudomonas spp., as demonstrated by phytotoxicity and antibiosis assays, as well as by red-blood-cell lysis. Differences in biological activity were putatively ascribed to its weak positive net charge at neutral pH. Planar lipid membrane experiments showed step-like current transitions, suggesting that cormycin is able to form pores. This ability was strongly influenced by the phospholipid composition of the membrane and, in particular, by the presence of sterols. All of these findings suggest that cormycin derivatives could find promising applications, either as antifungal compounds for topical use or as post-harvest biocontrol agents.

Aminoacylase 1 is a zinc-binding metalloprotease catalyzing the hydrolysis of N(alpha)-acylated l-amino acids; it presents altered expression levels in different renal and small cell lung carcinomas. A description of its redox and oligomerization state was achieved by combined biochemical and mass spectrometric procedures. A topological analysis of the enzyme structural architecture was derived from limited proteolysis and selective chemical modification experiments, using a broad range of proteases and chemical reagents. The analysis of the reaction products by different mass spectrometric techniques identified 26 amino acids as being accessible on the molecular surface, defining polypeptide regions exposed in the structure of the dimeric protein. The nature of the intermolecular contact zone between monomers was investigated by cross-linking reaction and mass mapping experiments. The cross-linked dimer was isolated, and the intermolecular cross-linked peptides were characterized, thus demonstrating the spatial proximity of Lys220 and Lys231 at the dimerization interface. Standard modeling procedures based on automatic alignment on the structure of members of the M20 peptidase family failed to produce a dimeric model consistent with experimental data. Discrepancies were observed mainly at the dimer interface and at loop regions. Therefore, a refined model for this dimeric protease was calculated by selecting the one able to generate a structure fully compatible with experimental findings, among all possible suboptimal sequence alignments. According to this model, each aminoacylase monomer consists of two domains: a globular catalytic subunit (residues 1-188 and 311-399) consisting of a beta-sheet sandwiched between alpha-helices and a second beta-sheet located on the surface, and the dimerization domain (residues 189-310) folding into a beta-sheet flanked on one side by two alpha-helices. These results indicate that reliable approaches such as limited proteolysis, selective chemical modification, and cross-linking coupled to mass spectrometry can be used to test and optimize molecular models of multimeric proteins and highlight problems in automatic model building.

The conformational features of human melanin-concentrating hormone (hMCH) [Asp1-Phe2-Asp3-Met4-Leu5-Arg6-cyclo(S[bond]S)(Cys7-Met8-Leu9-Gly10-Arg11-Val12-Tyr13-Arg14-Pro15-Cys16)-Trp17-Gln18-Val19], in water and in a CD(3)CN/H(2)O (1:1 v/v) mixture at 298 K, have been determined by NMR spectroscopy followed by simulated annealing and molecular dynamics analyses to identify conformer populations. Backbone clustering analysis of NMR-spectroscopy-derived structures in the 7-16 peptide region led to the identification of a single representative structure in each solvent. Both root mean square deviation clustering and secondary structure analysis of the final conformers in both solvents show substantial convergence of most conformers into a single fold in the 4-17 region, with a limited variability around Gly10 and Tyr13 on going from CD(3)CN/H(2)O to pure water. The main feature deduced from the analysis of secondary structures is the occurrence of an N-terminal alpha helix of variable length, which spans an overall residue range of 2-9. A comparative analysis in the two solvents highlights that these structures are substantially different from that reported in the literature for the cyclic MCH(5-14) subunit of salmon MCH, which was used to perform a molecular characterization of the MCH/receptor complex. Our conformational data call for a critical revision of the proposed MCH/receptor complex model.

The objective of our study was to mimic in a typical reductionist approach the molecular interactions observed at the interface between the gp130 receptor and interleukin-6 during formation of their complex. A peptide system obtained by reproducing some of the interleukin-6/gp130 molecular interactions into a two-helix bundle structure was investigated. The solution conformational features of this system were determined by CD and NMR techniques. The CD titration experiments demonstrated that the interaction between the designed peptides is specific and based on a well-defined stoichiometry. The NMR data confirmed some of the structural features of the binding mechanism as predicted by the rational design and indicated that under our conditions the recognition specificity and affinity can be explained by the formation of a two-helix bundle. Thus, the data reported herein represent a promising indication on how to develop new peptides able to interfere with formation of the interleukin-6/gp130 complex. Copyright  2003 European Peptide Society and John Wiley & Sons, Ltd.

The achiral, nitroxyl-containing alpha-amino acid TOAC (TOAC = 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), in combination with the chiral alpha-amino acid C(alpha)-methyl valine [(alphaMe)Val], was used to prepare short peptides (from di- to hexa-) that induced the enantioselective oxidation of racemic 1-phenylethanol to acetophenone. The best catalyst was an N(alpha)-acylated dipeptide alkylamide with the -TOAC-(alphaMe)Val- sequence folded in a stable, intramolecularly hydrogen-bonded beta-turn conformation with large, lipophilic (hydrophobic) N- and C-terminal blocking groups. We rationalized our findings by proposing models for the diastereomeric intermediates between (R)-[and (S)]-1-phenylethanol and the catalyst Fmoc-TOAC-L-(alphaMe)Val-NHiPr, based on the X-ray diffraction structure of the latter.

A complete series of Nα-protected, monodispersed homooligopeptide esters to the pentamer level from L-Cα-methyl,Cα-allylglycine (L-Mag) have been synthesized step-by-step in solution and fully characterized. The solution preferred conformation of these homooligomers has been assessed by FT-IR absorption and 1H NMR techniques. Moreover, the molecular structures of the homedimer and trimer have been determined in the crystal state by X-ray diffraction, and the conformational energy map of the homotrimer has been computed. The results obtained point to the conclusion that right-handed, single, or multiple β-bends are preferentially adopted by the conformationally restricted L-Mag homooligomers. In particular, 310-helices are formed by the longest homooligomer (pleionomer). The implications for the use of the Mag residue in designing conformationally constrained peptide substrates for reactions involving the side-chain C=C functionality are briefly discussed.

Recently, Verdine and coworkers experimentally investigated the olefin side-chain to side-chain ring-closing metathesis (RCM) reaction in α-helical peptides with two allyl-based, Cα-methylated α-amino acids of various side-chain length incorporated at the i, i+4 and i, i+7 positions [1], Here we describe our theoretical and experimental findings on the extension of the Verdine’s approach to β-turn/310-helical peptides where the allyl-based, Cα-methylated α-amino acids are inserted at the i, i+3 positions.

The interaction between the chemokine receptor CXCR4 and its ligand, CXCL12, has been shown in pre-clinical models to facilitate tumour metastasis and to be involved in tissue repair and in recruitment of hematopoietic precursors. AMD3100, a potent CXCR4 antagonist, has been approved by FDA for stem cell mobilization in patients with multiple myeloma and non-Hodgkin lymphoma. Several other CXCR4 inhibitors are in clinical development (BTK140, CTCE-9908, MDX-1338 and POL6326), with the aim of inhibiting the development and progression of metastasis. In the rational design of CXCR4 inhibitors, a new class of cyclic peptide CXCL12-based CXCR4 inhibitors was discovered (IT Patent M12010A 000096; PCT WO2011/092575 A1) and validated for efficacy in in vitro and in vivo models. Among the tested peptides RFF-1 showed potent and specific inhibitory effects on CXCR4-mediated functions in multiple in vitro assays (P-ERK induction, migration and calcium mobilization CXCL12-induced and binding to CXCR4). C57/BL mice were injected with B16 melanoma cells transfected with human CXCR4. A 4,5 fold reduction in lung metastases was observed after 10 days treatment with 2 mg/kg intraperitoneally (i.p.) RFF-1 compared to the CXCR4 inhibitor AMD3100 (1.25mg/kg i.p), the powerful CXCR4 inhibitor not suitable for prolonged administration, that reduced lung metastases of about 4 fold. In another model of mouse osteosarcoma, K7M2 murine cells, were injected in Balb/C mice. A 3 fold reduction in lung metastases was observed after 15 days treatment with 10 mg/kg i.p. RFF-1 whereas AMD3100 treatment (2.5mg/kg i.p.) lead to 1.8 fold increase in lung metastases. Additionally, a 30% reduction in primary cell growth was achieved in a subcutaneous model of human renal cancer SN12C cells when i.p. treated with 2 mg/kg RFF-1. With the aim to evaluate RFF-1 efficacy in hematopoietic stem cell (HSC) mobilization DBA/2 healthy mice were injected with a single s.c. RFF-1 dose and peripheral blood HSC mobilization was evaluated by colony forming unit assay. Treatment with 30 mg/kg RFF-1 results in rapid, potent and durable mobilization of HSC reaching faster the peak compared to 20 mg/kg AMD3100. These results have prompted us to pursue the clinical development of peptide RFF-1. A first in human Phase I trial is planned to determine the maximal tolerated dose and recommended Phase 2 dose, toxicity profile, pharmacokinetics and antitumor activity of RFF-1 in patients with refractory solid tumors. The Phase I-II study of a novel CXCR4 antagonist provides the opportunity to develop settings in diseases where there is increasing evidence of a role for CXCR4 in the development and progression of metastases such as relapsed glioblastoma, neoadjuvant colorectal and breast cancer, and advanced prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4645. doi:1538-7445.AM2012-4645

Introduction: The CXCR4 inhibitor AMD3100 is FDA-approved to provide stem cell mobilization for autologous transplantation in NHL and Multiple Myeloma patients. Newly designed CXCR4 inhibitory molecules, named Hys-7 inv, Phe-7 inv and Hys-OH, were recently discovered and validated in in vitro and in vivo studies (patent MI2010A-000093). Aim of the study was to evaluate the role of new CXCR4 inhibitors in hematopoietic stem cell and neutrophil mobilization. Experimental procedures: 60 DBA/2 female were injected subcutaneously with 5 or 30 mg/kg of CXCR4 antagonist in comparison with AMD3100 (3 or 20 mg/kg). Blood was collected at 1, 2, 6 and 24 hours from the treatment. Neutrophilis were evaluated trough flow cytometry (Ly-6G+ cells) and colony forming unit assay (CFU) of the mobilized stem cell were performed. Bone marrow was also evaluated through flow cytometry and cytological analysis. Results: Hys-7 inv, Phe-7 inv and Hys-OH, cyclic peptides CXCR4 antagonists, induced potent stem cell and neutrophils mobilization compared with AMD3100. Treatment resulted in a rapid and dose related increment in neutrophils count as well as hematopoietic stem and progenitor cells increase in peripheral blood (2 to 5 fold increase). Although Hys-7inv and Hys-OH induced neutrophils increment similar to AMD3100 the kinetic was different. AMD3100 (20 mg/kg) mobilized Ly-6G cells within 1 hour but transiently (no increase at 24 hours); instead Hys-7inv and Hys-OH mobilized Ly-6G within 2 hours lasting up to 24 hours. Interestingly, Phe-7 inv increased neutrophils mobilization within 1 hour lasting up to 24 hours. AMD3100 treated bone marrow showed an increase in progenitor cells myelo-committed (from 1.5 to 3 fold increase) and polimorphonuclear cells (PMN) (1.5 to 2.5 fold increase) and a reduction in mature-lymphocyte (0.4 to 0.28 fold increase); CXCR4 inhibitor peptides increased the immature myelo-committed progenitor cells (from 2 to 3 fold increase VS control) compared with AMD3100 (1.5 fold increase VS control) reducing PMN (1.5 to 1.65 fold increase) compared to AMD3100 (2.5 fold increase). CFU showed increased colonies in peripheral blood from mice treated with cyclic-peptides (from 2.5 to 4 fold increase) compared to AMD3100 (3.5 fold increase); the highest number of CFU was reached earlier in mice treated with Hys-7inv, Phe-7 inv and Hys-OH rapidly (within 1h) versus AMD3100 (6 hours). Conclusions: In vivo studies demonstrated that three newly synthesized CXCR4 inhibitors increased neutrophil and hematopoietic stem cells release from the bone marrow with different kinetic and duration compared to the FDA approved stem cell mobilizer AMD3100. Ongoing experiments are evaluating advantages in mobilizing precursors cells with CXCR4 cyclic-peptides antagonists compared to AMD3100. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2011-394

Comparative molecular dynamics simulations of both subtypes 1 and 2 of the melanin-concentrating hormone receptor (MCHR1 and MCHR2, respectively) in their free and hormone-bound forms have been carried out. The hormone has been used in its full-length and truncated forms, as well as in 16 mutated forms. Moreover, MCHR1 has been simulated in complex with T-226296, a novel orally active and selective antagonist. The comparative analysis of an extended number of receptor configurations suggests that the differences between inactive (i.e., free and antagonist-bound) and active (i.e., agonist-bound) states of MCHRs involve the receptor portions close to the E/DRY and NPxxY motifs, with prominence to the cytosolic extensions of helices 2, 3, 6, and 7. In fact, the active forms of these receptors share the release of selected intramolecular interactions found in the inactive forms, such as that between R3.50 of the E/DRY motif and D2.40, and that between Y7.53 of the NPxxY motif and F7.60. Another feature of the active forms of both MCHRs is the approach of “helix 8” to the cytosolic extension of helix 3. These features of the active forms are concurrent with the opening of a cleft at the cytosolic end of the helix bundle. For both MCHRs, the agonist-induced chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 6, following the ligand interaction with selected amino acids in the extracellular half of the receptor. Proteins 2004. © 2004 Wiley-Liss, Inc.