Dimer Bands Research Articles

Cryo-EM structures of the human band 3 transporter indicate a transport mechanism involving the coupled movement of chloride and bicarbonate ions.

The band 3 transporter is a critical integral membrane protein of the red blood cell (RBC), as it is responsible for catalyzing the exchange of bicarbonate and chloride anions across the plasma membrane. To elucidate the structural mechanism of the band 3 transporter, detergent solubilized human ghost membrane reconstituted in nanodiscs was applied to a cryo-EM holey carbon grid to define its composition. With this approach, we identified and determined structural information of the human band 3 transporter. Here, we present 5 different cryo-EM structures of the transmembrane domain of dimeric band 3, either alone or bound with chloride or bicarbonate. Interestingly, we observed that human band 3 can form both symmetric and asymmetric dimers with a different combination of outward-facing (OF) and inward-facing (IF) states. These structures also allow us to obtain the first model of a human band 3 molecule at the IF conformation. Based on the structural data of these dimers, we propose a model of ion transport that is in favor of the elevator-type mechanism.

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

Hydrogen bond complexation of ethanol with chlorobenzene: FTIR studies

ABSTRACT FTIR spectral studies has been carried out on ethanol (EtOH), chlorobenzene (PhCl) and their binary solutions at various EtOH/PhCl mole fractions. The bonded O − H stretching region has been deconvoluted into four Gaussian peaks centring at 3543.6, 3489.4, 3406.3 and 3254.3 cm−1. The first and the last peaks have been assigned to the hydroxyl stretching bands of dimer and tetramer, while the remaining two to the trimer bands. The presence of dimer in liquid PhCl has been confirmed by the experimental C − Cl stretching bands. Experimentally, two C − Cl stretching bands have been observed, which fall outside the 1096–1089 cm−1 range reported for chlorobenzenes in literature. The positional changes in O − H , C H 3 , C H 2 , C − H and C − Cl stretching bands in the spectrum of the solutions have been analysed in terms of heteromolecular H-bond interactions. This analysis shows that the classical O − H ⋯ Cl hydrogen bonds are the dominant interaction forces.

Solvent effect on the self-association of the 1,2,4-triazole: A DFT study

The self-association of 1,2,4-triazole was studied by the DFT method at the B3LYP/6-311++G(d,p) level in the gas phase, and the most stable dimer and trimer were presented. Based on the integral equation formalism polarizable continuum model (IEFPCM), the effect of solvents such as water and dimethyl sulfoxide (DMSO) on the geometric parameters of molecular complexes (dimer and trimer), atomic charge distribution, molecular electrostatic potential (MEP) surface, and frontier molecular orbitals (FMO) was analyzed. Intermolecular interactions in cyclic and linear complexes were studied using topological analyses (QTAIM, NCI, RDG, ELF, and LOL). IR spectra of 1,2,4-triazole monomer, dimer, and trimer were simulated and compared with experimental results. In self-association, intermolecular hydrogen bonding causes a red shift of the N–H stretching vibrational frequency of 1,2,4-triazole. The solvent effect on the vibrational bands of monomer, dimer, and trimer was analysed.

Расчет структуры и инфракрасных спектров поглощения водородно связанных комплексов метилформиата с фтористым водородом

Equilibrium nuclear configurations of three dimers formed by the most stable conformer of a methyl formate molecule with a hydrogen fluoride molecule are calculated using the MP2/aug-cc-pVTZ quantum–chemical method and taking the basis set superposition error into account. The frequencies and intensities for the IR absorption bands of dimers are determined both in the harmonic and anharmonic approximations with the use of the second-order perturbation theory. The calculation method was tested in a calculation of the spectrum of an isolated methyl formate molecule and by comparison with experimental data. The changes in the geometrical parameters of monomers and the charges on atoms under the action of hydrogen bonds were analyzed. The shifts of vibrational frequencies of the monomers upon formation of dimers were calculated. The strongest absorption bands of dimers that can be used in detection of these complexes in spectroscopic experiments were determined.

Trimers formed by formaldehyde with hydrogen fluoride: Structures, energetics, and infrared absorption spectra

Equilibrium nuclear configurations of two stable H2CO∙∙∙(HF)2 trimers and four stable (H2CO)2∙∙∙HF trimers are calculated in the MP2/aug-cc-pVTZ approximation with the basis set superposition error taken into account. Anharmonic values of frequencies and intensities for infrared absorption bands are computed. For comparison, the analogous calculations were carried out in the same approximation for the monomers and homo- and heterodimers. The changes in the spectral parameters of absorption bands upon formation of trimers are examined. The main emphasis was on revealing the cooperativity of H-bonding effects in trimers of different composition and the correlations between the intramonomer and intermonomer charge transfer, the structural and spectral changes upon formation of trimers. It was found that three predicted trimers have sufficiently large binding energies and their HF stretching bands are significantly shifted from strong bands of monomers and dimers, which can facilitate their experimental observation.

Oligomerization of the HBV/HDV functional receptor NTCP expressed in Sf9 insect cell

BackgroundSodium taurocholate co-transportering polypeptide (NTCP, SLC10A1) is a vital bile acid transporter and the functional receptor of hepatitis B and D virus. The oligomerization of NTCP is important for the structural study of its interaction with HBV preS1 peptide. MethodsRecombinant NTCPs were expressed in Sf9 host cell using baculoviruses. Function of recombinant NTCP was verified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. A quantitative fluorescence resonance energy transfer (FRET) method was established to analyze the interaction between NTCP wild type (WT) and mutants. Co-immunoprecipitation (Co-IP) was used to test the interaction between NTCP variants. ResultsSub-cellular location of recombinant NTCPs varies with the modification of NTCP. Bands of monomer, dimer and oligomers were shown in gel analysis of NTCP. Significant FRET was observed between cyan florescence protein (CFP) tagged NTCP and yellow florescence protein (YFP) tagged NTCP. FRET efficiency between CFP- and YFP-NTCP S267F mutants was lower than WT. Co-IP results showed that S267F interacts with WT NTCP when co-expressed in cell. ConclusionDimer is the predominant form of NTCP expressed in Sf9 when solubilized with detergent. FRET and Co-IP analysis support that NTCP forms oligomers in Sf9 cell. General significanceOur results showed that NTCP formed oligomers in Sf9 cell. Meanwhile the FRET analysis of NTCP variants further elucidated the molecular mechanism of NTCP oligomerization.

Effect of the TiO2 surface modification with 3-glycidyloxypropyltrimethoxysilane on the aggregation of cresyl violet: Application to a dye-sensitized solar cell

Dye aggregation is a typical phenomenon occurring in dye-sensitized solar cells (DSSCs), which causes a decrease in the electron injection efficiency from the photoexcited dye to titanium dioxide (TiO2). As this results in a decrease in the photovoltaic performance, it cannot be ignored in the study of DSSCs. This paper reports a DSSC involving a TiO2 electrode chemically modified by cresyl violet (CV) covalently attached to 3-glycidyloxypropyltrimethoxysilane (GPTMS). The aim of the study is to understand the influence of silane coupling agents (SCAs) on dye aggregation. Aggregation of CV occurring on the TiO2 surface was prevented by GPTMS treatment on the TiO2 surface. This result was revealed by the decreased absorption intensity of the dimeric band observed in the total transmittance spectra. Density functional theory calculations and fluorescence decay curve measurements indicated that the CV covalently attached to GPTMS acts as a photosensitizing dye in DSSCs. As a result, the photovoltaic performance of DSSCs containing CV was enhanced by preventing its aggregation upon GPTMS treatment onto the TiO2 surface (7-fold increase: 0.0020%–0.014%). We concluded that TiO2 surfaces treated with SCAs can help to prevent dye aggregation, and therefore, enhance the photovoltaic performance of DSSCs.

M-Protein Sequencing and Monitoring in Serum of LC-Only Multiple Myeloma Patients

M-Protein Sequencing and Monitoring in Serum of LC-Only Multiple Myeloma Patients

Fatty Acids and Their Metal Salts: A Review of Their Infrared Spectra in Light of Their Presence in Cultural Heritage.

In a cultural heritage context, fatty acids are usually found as breakdown products of lipid-containing organic remains in archaeological findings, binders in aged oil paintings, and additives in modern art-related materials. They may further interact with the ionic environment transforming into metal soaps, a process that has been recognized as a threat in aged paintings but has received less attention in archaeological objects. The investigation of the above related categories of materials with infrared spectroscopy can provide an overall picture of the organic components’ identity and demonstrate their condition and prehistory. The capability of investigating and distinguishing fatty acids and their metal soaps through their rich infrared features, such as the acidic carbonyl, the carboxylate shifts, the variable splits of alkyl chain stretching, bending, twisting, wagging, and rocking vibrations, as well as the hydroxyl peak envelopes and acid dimer bands, allows for their direct detailed characterization. This paper reviews the infrared spectra of selected saturated fatty monoacids and diacids, and their corresponding sodium, calcium, and zinc salts and, supported by newly recorded data, highlights the significance of their spectroscopic features.

Molecular and Polymeric Cadmium Nitrate Complexes with Bridging 3-Aminoquinoline: Synthesis, Structures, and Luminescence Properties

The reaction of Cd(NO3)2⋅4H2O with 3-aminoquinoline (3-Аq) affords the molecular and poly-meric complexes [Cd(NO3)2(H2O)(3-Аq)]⋅2MeCN (I) and {[Cd(NO3)2(3-Аq)2]⋅MeCN}n (II), respectively, depending on the ratio of the reagents and composition of the medium. Compounds I and II are studied by elemental analysis, X-ray structure analysis (CIF files CCDC nos. 2015059 (I) and 2015060 (II)), X-ray diffraction analysis, and luminescence investigation. In both cases, the environment of the cadmium atom is a pentagonal bipyramid. The 3-Аq molecules of dimer I are bridging ligands, and the monodentate mode of binding with Cd(II) along with the indicated function is observed in the polymer. Hydrogen bonds between the H atoms of the NH2 group and the N atom of the solvate MeCN molecule and the O atom of the NO3 group result in the formation of a three-dimensional supramolecular network, which is additionally strengthened by intermolecular stacking interactions between the aromatic fragments of 3-Аq of the adjacent molecules. Both compounds luminesce in the red spectral range. As compared to the spectrum of free 3-Аq, the shift of the emission bands of dimer I and polymer II in this range is 93 and 38 nm, respectively.

CC-stretched formic acid: isomerisation, dimerisation, and carboxylic acid complexation.

The cis-trans-isomerism of the propiolic acid monomer (HC[triple bond, length as m-dash]C-COOH) is examined with linear Raman jet spectroscopy, yielding the first environment-free vibrational band centres of a higher-energy cis-rotamer beyond formic acid (HCOOH) in addition to all fundamentals and a large number of hot and combination/overtone bands of the trans-conformer. Two near-isoenergetic trans-fundamentals of different symmetry (CC[double bond, length as m-dash]O bend and OH torsion) prove to be a sensitive benchmarking target, as their energetic order is susceptible to the choice of electronic structure method, basis set size, and inclusion of vibrational anharmonicity. For the infrared- and Raman-active C[double bond, length as m-dash]O stretching fundamentals of the cyclic (C2h) trans-propiolic acid dimer, resonance couplings are found that in part extend to the Cs-symmetric heterodimer of trans-propiolic and trans-formic acid. Exploratory vibrational perturbation theory (VPT2) calculations show that all perturbing states involve displacements of the OH moieties located on the doubly hydrogen bonded ring. The comparison of the infrared spectra of the propiolic acid dimer and its heterodimer with formic acid to that of several other carboxylic acid dimers from the literature reveals a notable similarity regarding a non-fundamental dimer band around 1800 cm-1, which in most cases is so far unassigned. VPT2 calculations and a simple harmonic model suggest an assignment to a combination vibration of the symmetric and antisymmetric OH torsion.

Abstract 472: What electric field strength is necessary for maximum tumor-treating fields efficacy

Abstract Why Is 4 V/cm Electric Field Strength Necessary for Maximum Tumor-Treating Fields Efficacy? INTRODUCTION: Tumor Treating Fields (TTFields) extend overall survival in glioblastoma patients. Threshold field strength for TTFields is typically cited as 2V/cm. Yet plots of cell death vs. field strength in vitro indicate a power law relationship asymptotic to 4 V/cm for 100% efficacy. The mechanisms of action (MoA) of TTFields underlying this relationship are currently under investigation. TTFields are suspected of disrupting critical mitotic processes performed by large polar cellular molecules such as microtubules (MT). METHODS: Using the power law relationship for the energy (intensity, I) carried by an electric field and its amplitude (Eq. 1), we calculated energy vs. TTFields amplitude. (1) RESULTS: We suggest Eq. 1 is a likely source of the relationship between TTFields' amplitude and cell death proportion that has been demonstrated empirically. Further, Table 1 shows energy transmitted per unit MT surface area per TTFields cycle, specifically, the energy per MT dimer band around an MT helix per TTFields period of 5 µs. TTFields Field Strength (V/cm)Radiant Flux Density (W/nm2)Energy/(Cycle-Area) (J/nm2)Energy/MT Dimer/Cycle (J)Energy/MT Dimer Band/Cycle (J)113.3 × 10−186.63 × 10−234.01 × 10−224.17 × 10−20253.1 × 10−1826.6 × 10−231.60 × 10−211.67 × 10−193119 × 10−1859.7 × 10−233.61 × 10−213.75 × 10−194212 × 10−18106 × 10−236.42 × 10−216.67 × 10−195332 × 10−18166 × 10−231.00 × 10−201.04 × 10−186478 × 10−18239 × 10−231.44 × 10−201.50 × 10−18 A metric for disruption of cellular processes is 1-2 orders of magnitude greater than the thermal background energy in the cell, kT = 4.3 × 10−21 J. Table 1 shows that the energy absorbed by a MT dimer is insufficient to disrupt cellular processes at 2 V/cm, while due to energy increasing with square of field strength, at 5 V/cm it may be disruptive. For an entire dimer band around the MT helix, 2 V/cm appears to be sufficient, while 4 V/cm seems amply strong to disrupt MT function, in accordance with in vitro experiments. CONCLUSION: Assuming the power law of energy vs. electric field strength and the resulting energy absorbed by microtubule components, we found a correlation between typical TTFields' amplitudes and estimated disruption thresholds of MT function. TTFields MoA. 4 V/cm, not 2V/cm, should be the target field strength to realize the full efficacy of TTFields. Citation Format: Kristen W. Carlson, Jack A. Tuszynski, Socrates Dokos, Nirmal Paudel, Zeev Bomzon. What electric field strength is necessary for maximum tumor-treating fields efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 472.

Abstract 5393: Association of heat shock proteins as chaperone for STING: A potential link in a key immune activation mechanism revealed by the novel anti-cancer agent PV-10

Abstract Introduction: The activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor system located in the endoplasmic reticulum, has been shown to augment antitumor immunity through the induction of pro-inflammatory cytokines. Currently, a number of STING agonists have been developed to treat refractory malignancies. Our previous studies have identified PV-10 (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) as a novel therapeutic agent with potent activity following intra-tumoral injection. Here we describe a previously unidentified mechanism by which PV-10 may facilitate sustained immune activation and therapeutic antitumor activity. Methods: The well-established acute monocytic leukemia (AML) cell line THP-1 was used as a model to study STING activation in vitro. Cells were treated with PV-10 and the induction of STING was evaluated by Western blot analysis using cGAMP as a positive control. Proteins that associate with STING in the presence of PV-10 were purified by immunoprecipitation and analyzed by mass spectrometry (LC-MS/MS, Mascot database). The culture supernatants from PV-10 treated cells were probed for a panel of 42 immune cytokines using the Bio-Plex multiplex bead-based assay system. Results: We show that the exposure of THP-1 cells to PV-10 leads to the appearance of a new 70-KD STING dimer band detected by specific antibodies. Compared to cGAMP controls no induction of PDL-1 was noted. Mass spectrometric analysis of immuno-precipitates of STING in these cells showed the presence of Heat Shock Proteins (HSP) 60, 70 and 90 as well as Polyadenylate Binding Protein 1 (PABP1) to the dimerized STING complex. The chemokine assays showed specific upregulation of a distinct set of pro-inflammatory and cytotoxic T-cell recruitment cytokines. A peak in the induction of MCP-3 and IFN gamma was seen at 24 hours (2 fold) and an approximately 10-fold increase in IL-6, IL-8 and IP-10 was seen in the 24 hours following exposure to PV-10. A significant increase in MCP-1 levels was also noted. Discussion: The compound PV-10 has been shown to induce tumor necrosis following intra-tumoral injection. Our present data show a possible mechanism of this agent through STING dimerization and HSP association leading to a sustained pro-inflammatory and immune response. We provide experimental data, for the first time, for a role of HSPs in STING-mediated immune activation pathways and the description of an agent that may play a role in effective single-agent immunotherapy or drug combination therapy approaches in future clinical studies. Citation Format: Satbir Thakur, Chunfen Zhang, Laurent Brechenmacher, Luis Murgia-Favela, Aru Narendran. Association of heat shock proteins as chaperone for STING: A potential link in a key immune activation mechanism revealed by the novel anti-cancer agent PV-10 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5393.

Electric Field Effect on Condensed-Phase Molecular Systems. X. Interconversion Dynamics and Vibrational Stark Effect of Hydrogen Chloride Clusters in an Argon Matrix.

In this study, the effect of a strong (≤4 × 108 V·m-1) dc electric field on hydrogen chloride (HCl) dimers and trimers isolated in a solid argon matrix has been investigated using the ice film nanocapacitor and reflection-absorption infrared spectroscopy methods. The H-Cl vibrational bands of the HCl dimers showed a linear Stark frequency shift and an increased intensity under the applied electric field, and these changes were reversible with the electric field strength. This behavior indicated that the dimers were reoriented by the applied electric field. The reorientation occurred via tunneling inversion of individual HCl subunits of the dimer, which interconverted the proton-accepting and -donating HCl subunits, as observed for the heterodimers HCl-DCl and DCl-HCl. The interconversion of dimers could occur even at low electric field strength (∼107 V·m-1) and was almost complete above the field strength of 1.0 × 108 V·m-1. In contrast, the asymmetric H-Cl stretching bands of the HCl trimers exhibited Stark broadening under the influence of the electric field without a shift in frequency or change in intensity. This behavior indicated that the cyclic structure of the HCl trimer was stable even when subjected to a strong electric field. The Stark sensitivity factor (Δμ) of H-Cl vibrations was deduced from the Stark effect analysis of the HCl dimer and trimer bands, which gave the following: ΔμD1 = 2.3 ± 0.2 cm-1/(108 V·m-1) for the proton-acceptor subunit of the dimer, ΔμD2 = 5.1 ± 0.5 cm-1/(108 V·m-1) for the proton-donor subunit of the dimer, and ΔμT = 4.5 ± 0.5 cm-1/(108 V·m-1) for the asymmetric stretching vibration of the cyclic trimer.

Coherent intradimer dynamics in reaction centers of photosynthetic green bacterium Chloroflexus aurantiacus

Early-time dynamics of absorbance changes (light minus dark) in the long-wavelength Qy absorption band of bacteriochlorophyll dimer P of isolated reaction centers (RCs) from thermophilic green bacterium Chloroflexus (Cfx.) aurantiacus was studied by difference pump-probe spectroscopy with 18-fs resolution at cryogenic temperature. It was found that the stimulated emission spectrum gradually moves to the red on the ~100-fs time scale and subsequently oscillates with a major frequency of ~140 cm−1. By applying the non-secular Redfield theory and linear susceptibility theory, the coherent dynamics of the stimulated emission from the excited state of the primary electron donor, bacteriochlorophyll dimer P*, was modeled. The model showed the possibility of an extremely fast transition from the locally excited state P1* to the spectrally different excited state P2*. This transition is clearly seen in the kinetics of the stimulated emission at 880 and 945 nm, where mostly P1* and P2* states emit, respectively. These findings are similar to those obtained previously in RCs of the purple bacterium Rhodobacter (Rba.) sphaeroides. The assumption about the existence of the second excited state P2* helps to explain the complicated temporal behavior of the ΔA spectrum measured by pump-probe spectroscopy. It is interesting that, in spite of the strong coupling between the P1* and P2* states assumed in our model, the form of the coherent oscillations is mainly defined by pure vibrational coherence in the excited states. A possible nature of the P2* state is discussed.

Spectrophotometric-chemometrics study of the effect of solvent composition and temperature on the spectral shape and shift of copper and nickel phthalocyanines in different aqueous-nonaqueous mixed solvents.

Phthalocyanines (Pcs) are green-blue colored aromatic macrocyclic compounds used extensively in the dyeing industry. The assembly phenomenon for dye molecules is directly traceable by most of the spectroscopic methods. In this investigation, the monomer-dimer equilibria of copper and nickel phthalocyanines tetra sulfonic acid tetrasodium salts (CuPcTS, NiPcTS) have been investigated by spectrophotometric and chemometrics methods in binary mixtures of H2O-DMSO and H2O-CH3CN. The dimerization constants, (KD), enthalpies and entropies of CuPcTS and NiPcTS have been calculated by studying the UV-Vis spectra at different concentrations of dyes (10-6 to 10-4molL) and in the temperature range 298-343 K and in some samples up to 353 K by multivariate curve resolution (mcr) methods. By increasing the temperature, the value ofKD decreases. The inverse temperature dependence of KD(van't Hoff equation) was used for determination of ΔH0 and ΔS0and following that ΔG0of the dimerization reactions. As a result, upon aggregation, an increase in the intensity of the new shoulder at (~600 nm) and the Q-band at (662-670 nm) and concomitant decrease of the dimer band at (630-624 nm) are observed for all of the samples in different solvents composition. Therefore, the H-dimer type of these pigments was notable, in studied binary solvents. The effect of the solvent composition, concentration dye, and temperature on the spectral responses, the exciton parameters and concentration distribution diagrams of the two pigments were studied and discussed. The density functional theory (DFT) calculations were done in the aqueous and gas phase that they were included the HOMO-LUMO energies and the simulated UV-Vis spectrum. These calculations were beneficial for studying the UV-Vis spectrum of it in the aqueous phase for checking experimental data.

Abstract LB-114: RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF

Abstract RAS mutant tumors have proven largely refractory to inhibitors of the MAPK pathway in clinic. In contrast, selective inhibitors of BRAFV600E but not wild type RAF have been successfully developed in BRAFV600E mutant melanoma, NSCLC, and thyroid cancers. Success of these drugs results largely from the therapeutic index imparted by lack of activity against wild RAFs; however, this selectivity also prevents their utility in indications beyond BRAFV600E disease, such as RAS-mutant tumors. In contrast, MEK1/2 inhibitors, while effective pre-clinically in RAS mutant models, have proven ineffective against RAS mutant tumors due at least in part to the poor therapeutic index of these molecules that results from pathway inhibition in normal cells. Thus, to inhibit effectively MAPK pathway signaling in tumors, inhibitors with improved tolerability via differential pathway inhibition in tumor vs normal tissues are required. Recent data in mice conditionally lacking different RAF paralogs suggest that inhibitors biased towards CRAF might retain anti-tumor properties, with improved tolerability. LXH254 is a RAF inhibitor with high selectivity and nanomolar potency for BRAF and CRAF. Profiling of LXH254 activity in Ras mutant lines lacking either ARAF, BRAF or CRAF by CRISPR-Cas9 modification revealed that loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. This increased sensitivity to LXH254 from ARAF knockout was rescued by over-expression of wild-type ARAF but not by either ARAF that harbored mutations that impaired kinase activity (K336M, D447A) or its ability to dimerize (R362H). Comparing the effect of LXH254 on MAPK pathway signaling in RAS mutant cells engineered to express only one of the three RAF paralogs indicated that significantly higher concentrations of LXH254 are required to inhibit MAPK signaling in cells expressing only ARAF. Furthermore, at low concentrations of LXH254, cells expressing only ARAF displayed paradoxical activation of MAPK signaling in a manner similar to the BRAFV600E selective inhibitor dabrafenib. Thus, both LXH254 and dabrafenib inhibit mutationally activated monomeric BRAF, and promote/stabilize RAS-dependent dimerization of WT RAFs. However, because dabrafenib is only able to effectively inhibit one of the two RAF protomers in each dimer it paradoxically activates MAPK signaling in a RAS-dependent manner. In contrast, LXH254 retains activity against dimerized B-and C-RAF (but not ARAF) thereby theoretically extending its utility beyond BRAF mutant disease with better tolerability relative to, e.g. MEK1/2 inhibitors. Citation Format: Kelli-Ann Monaco, Scott Delach, Paul Fordjour, Salonee Parikh, Yun Feng, Mariela Jaskelioff, Darrin Stuart, Giordano Caponigro. RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-114.

Gel electrophoresis in combination with laser ablation-inductively coupled plasma mass spectrometry to quantify the interaction of cisplatin with human serum albumin.

Cisplatin and its second and third generation analogues are widely used in the treatment of cancer. To study their reactions with proteins, we present a method based on SDS-PAGE separation and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) for platinum detection in the reaction between human serum albumin (HSA) and cisplatin. We developed matrix-matched standards of HSA/cisplatin mixtures and used them to quantify the amount of adducts formed at different HSA:cisplatin ratios. We noted that cisplatin incubation with HSA resulted in the formation of higher order HSA n-mers, depending on the amount of cisplatin added. This caused a depletion of the HSA dimer bands, while the majority of HSA was present as the monomer. Inducing the formation of such higher molecular weight species may have an impact on the mode of action of metallodrugs.

Infrared bands of CS2 dimer and trimer at 4.5 μm.

We report observation of new infrared bands of (CS2)2 and (CS2)3 in the region of the CS2 ν1 + ν3 combination band (at 4.5 µm) using a quantum cascade laser. The complexes are formed in a pulsed supersonic slit-jet expansion of a gas mixture of carbon disulfide in helium. We have previously shown that the most stable isomer of (CS2)2 is a cross-shaped structure with D2d symmetry and that for (CS2)3 is a barrel-shaped structure with D3 symmetry. The dimer has one doubly degenerate infrared-active band in the ν1 + ν3 region of the CS2 monomer. This band is observed to have a rather small vibrational shift of -0.844 cm-1. We expect one parallel and one perpendicular infrared-active band for the trimer but observe two parallel bands and one perpendicular band. Much larger vibrational shifts of -8.953 cm-1 for the perpendicular band and -8.845 cm-1 and +16.681 cm-1 for the parallel bands are observed. Vibrational shifts and possible vibrational assignments, in the case of the parallel bands of the trimer, are discussed using group theoretical arguments.