Chemogenomic discovery of allosteric antagonists at the GPRC6A receptor

GPRC6A is a Family C G protein-coupled receptor recently discovered and deorphanized by our group. This study integrates chemogenomic ligand inference, homology modeling, compound synthesis, and pharmacological mechanism-of-action studies to disclose two noticeable results of methodological and pharmacological character: (1) chemogenomic lead identification through the first, to our knowledge, ligand inference between two different GPCR families, Families A and C; and (2) the discovery of the most selective GPRC6A allosteric antagonists discovered to date. The unprecedented inference of?pharmacological activity across GPCR families provides proof-of-concept for in?silico approaches against Family C targets based on Family A templates, greatly expanding the prospects of successful drug design and discovery. The antagonists were tested against a panel of seven Family A and C G protein-coupled receptors containing the chemogenomic binding sequence motif where some of the identified GPRC6A antagonists showed some activity. However, three compounds with at least ～3-fold selectivity for GPRC6A were discovered, which present a significant step forward compared with the previously published GPRC6A antagonists, calindol and NPS 2143, which both display ～30-fold selectivity for the calcium-sensing receptor compared to GPRC6A. The antagonists constitute novel research tools toward investigating the signaling mechanism of the GPRC6A receptor at the cellular level and serve as initial ligands for further optimization of potency and selectivity enabling future ex?vivo/in?vivo pharmacological studies.     

Increased fraction of low-density structures in aqueous solutions of fluoride

X-ray absorption spectroscopy (XAS) and small angle x-ray scattering (SAXS) were utilized to study the effect of fluoride (F(-)) anion in aqueous solutions. XAS spectra show that F(-) increases the number of strong H-bonds, likely between F(-) and water in the first hydration shell. SAXS data show a low-Q scattering intensity increase similar to the effect of a temperature decrease, suggesting an enhanced anomalous scattering behavior in F(-) solutions. Quantitative analysis revealed that fluoride solutions have larger correlation lengths than chloride solutions with the same cations but shorter compared to pure water. This is interpreted as an increased fraction of tetrahedral low-density structures in the solutions due to the presence of the F(-) ions, which act as nucleation centers replacing water in the H-bonding network and forming stronger H-bonds, but the presence of the cations restricts the extension of strong H-bonds.     

Insights into the Mechanism of Protein Electrospray Ionization From Salt Adduction Measurements

The mechanisms whereby protein ions are liberated from charged droplets during electrospray ionization (ESI) remain under investigation. Compact conformers electrosprayed from aqueous solution in positive ion mode likely follow the charged residue model (CRM), which envisions analyte release after solvent evaporation to dryness. The concentration of nonvolatile salts such as NaCl increases sharply within vanishing CRM droplets, promoting nonspecific pairing of Cl^- and Na⁺ with charged groups on the protein surface. For unfolded proteins, it has been proposed that ion formation occurs via the chain ejection model (CEM). During the CEM proteins are expelled from the droplet long before complete solvent evaporation has taken place. Here we examine whether salt adduction levels support the view that folded and unfolded proteins follow different ESI mechanisms. Solvent evaporation during the CEM is expected to be less extensive and, hence, the salt concentration at the point of protein release should be substantially lower than for the CRM. CEM ions should therefore exhibit lower adduction levels than CRM species. We explore the adduction behavior of several proteins that were chosen to allow comparative studies on folded and unfolded structures in the same solution. In-source activation eliminates chloride adducts via HCl release, generating protein ions that are heterogeneously charged because of sodiation and protonation. Sodiation levels measured under such conditions provide estimates of the salt adduction behavior experienced by the “nascent” analyte ions. Sodiation levels are significantly reduced for unfolded proteins, supporting the view that these species are indeed formed via the CEM.

Cation-Induced Stabilization of Protein Complexes in the Gas Phase: Mechanistic Insights From Hemoglobin Dissociation Studies

Collision-induced dissociation (CID) of electrosprayed protein complexes usually involves asymmetric charge partitioning, where a single unfolded chain gets ejected that carries a disproportionately large fraction of charge. Using hemoglobin (Hb) tetramers as model system, we confirm earlier reports that bound metal ions can stabilize protein complexes under CID conditions. We examine the mechanism underlying this effect. Nonvolatile salts cause extensive adduct formation. Significant stabilization was observed for Mg²⁺ and Ca²⁺, whereas K⁺, Rb⁺, and Cs⁺ had no effect. Precursor ion selection was used to examine Hb subpopulations with well-defined metal binding levels. K⁺, Rb⁺, and Cs⁺-adducted tetramers eject monomers that carry roughly one-quarter of the metal ions that were bound to the precursor. This demonstrates that charge migration during CID is exclusively due to proton transfer, not metal ion transfer. Also, replacement of highly mobile charge carriers (protons) with less mobile species (metal ions) does not exert a stabilizing influence under the conditions used here. Interestingly, Hb carrying stabilizing ions (Mg²⁺ and Ca²⁺) generates monomeric CID products that are metal depleted. This effect is attributed to a combination of two factors: (1) Me²⁺ binding stabilizes Hb via formation of chelation bridges (e.g., R-COO^– Me^{2+ –}OOC-R); the more Me²⁺ a subunit contains the more stable it is. (2) More than ~90 % of the tetramers contain at least one subunit with a below-average number of Me²⁺. The prevalence of monomeric CID products with depleted Me²⁺ levels is caused by the tendency of these low metal-containing subunits to undergo preferential unfolding/ejection.

Novel aspects of nanocellulose

Novel nanoscaled cellulose particles were prepared using high-pressure homogenization of aqueous media contenting treated cellulose samples in a Microfluidizer^® processor (MF). Here, we present the generation of spherical cellulose nanoparticles as an extension of previously published reports of nano fibrillated cellulose. Although MF treatment of unmodified cellulose yields nanofibrils which are reported in several publications, in the current work different kinds of pretreatments were proven to be necessary to obtain spherical structured cellulose nanoparticles. One such treatment may be the decrystallization of cellulose regenerating it from N-methylmorpholine-N-oxid-monohydrate (NMMNO*H₂O). Nanocellulose was then obtained by a subsequent high-pressure mechanical treatment of the precipitate in aqueous dispersion. Decrystallization was also realized by grinding cellulose in a planetary ball mill. The resulting amorphous intermediates were characterized by Raman spectroscopy. Another approach tested was hydrolysis and subsequent mechanical treatment using an Ultra-Turrax^® and MF. Another alternative was given by the mechanical treatment of aqueous dispersions of low substituted cellulose derivatives such as carboxymethyl cellulose and oxidized cellulose without any further hydrolysis.     

Regiospecific Formation and Unusual Optical Properties of 2,5‐Bis(arylethynyl)rhodacyclopentadienes: A New Class of Luminescent Organometallics

A series of 2,5‐bis(arylethynyl)rhodacyclopentadienes has been prepared by a rare example of regiospecific reductive coupling of 1,4‐(p‐R‐phenyl)‐1,3‐butadiynes (R?H, Me, OMe, SMe, NMe₂, CF₃, CO₂Me, CN, NO₂, ?C?C‐(p‐C₆H₄?NHex₂), ?C?C?(p‐C₆H₄?CO₂Oct)) at [RhX(PMe₃)₄] ( 1 ) (X=?C?C?SiMe₃ ( a ), ?C?C‐(p‐C₆H₄?NMe₂) ( b ), ?C?C?C?C?(p‐C₆H₄?NPh₂) ( c ) or ?C?C?{p‐C₆H₄‐C?C?(p‐C₆H₄‐N(C₆H₁₃)₂)} ( d ) or Me ( e )), giving the 2,5‐bis(arylethynyl) isomer exclusively. The rhodacyclopentadienes bearing a methyl ligand in the equatorial plane (compound 1 e ) have been converted into their chloro analogues by reaction with HCl etherate. The rhodacycles thus obtained are stable to air and moisture in the solid state and the acceptor‐substituted compounds are even stable to air and moisture in solution. The photophysical properties of the rhodacyclopentadienes are highly unusual in that they exhibit, exclusively, fluorescence between 500–800 nm from the S₁ state, with quantum yields of Φ=0.01–0.18 and short lifetimes (τ=0.45–8.20 ns). The triplet state formation (Φ_ISC=0.57 for 2 a ) is exceptionally slow, occurring on the nanosecond timescale. This is unexpected, because the Rh atom should normally facilitate intersystem crossing within femto‐ to picoseconds, leading to phosphorescence from the T₁ state. This work therefore highlights that in some transition‐metal complexes, the heavy atom can play a more subtle role in controlling the photophysical behavior than is commonly appreciated.     

Hydridoorganostannylene Coordination: Group 4 Metallocene Dichloride Reduction in Reaction with Organodihydridostannate Anions

Organodihydridoelement anions of germanium and tin were reacted with metallocene dichlorides of Group 4 metals Ti, Zr and Hf. The germate anion [Ar*GeH₂]⁻ reacts with hafnocene dichloride under formation of the substitution product [Cp₂Hf(GeH₂Ar*)₂]. Reaction of the organodihydridostannate with metallocene dichlorides affords the reduction products [Cp₂M(SnHAr*)₂] (M=Ti, Zr, Hf). Abstraction of a hydride substituent from the titanium bis(hydridoorganostannylene) complex results in formation of cation [Cp₂M(SnAr*)(SnHAr*)]⁺ exhibiting a short Ti–Sn interaction. (Ar*=2,6-Trip₂C₆H₃, Trip=2,4,6-triisopropylphenyl).     

Latest Advances Towards Ras Inhibition: A Medicinal Chemistry Perspective

Owing to their high occurrence rate across many human cancers and their lack of druggability so far, mutant forms of the signaling protein Ras are currently among the most attractive (and elusive) oncology targets. This strong appeal explains the sustained effort in the field, and the ensuing progress has rekindled optimism regarding the discovery of Ras inhibitors. In this Minireview, we discuss the most recent advances towards irreversible inhibitors, and highlight approaches to inhibitors of Ras–effector interactions that have been overshadowed by the current focus on direct Ras inhibition. At the same time, we provide a critical assessment from a medicinal chemistry perspective.     

Impact of the Spatial Organization of Bifunctional Metal–Zeolite Catalysts on the Hydroisomerization of Light Alkanes

Improving product selectivity by controlling the spatial organization of functional sites at the nanoscale is a critical challenge in bifunctional catalysis. We present a series of composite bifunctional catalysts consisting of one-dimensional zeolites (ZSM-22 and mordenite) and a γ-alumina binder, with platinum particles controllably deposited either on the alumina binder or inside the zeolite crystals. The hydroisomerization of n-heptane demonstrates that the catalysts with platinum particles on the binder, which separates platinum and acid sites at the nanoscale, leads to a higher yield of desired isomers than catalysts with platinum particles inside the zeolite crystals. Platinum particles within the zeolite crystals impose pronounced diffusion limitations on reaction intermediates, which leads to secondary cracking reactions, especially for catalysts with narrow micropores or large zeolite crystals. These findings extend the understanding of the “intimacy criterion” for the rational design of bifunctional catalysts for the conversion of low-molecular-weight reactants.     

Stereoselective Oxidative Bioconjugation of Amino Acids and Oligopeptides to Aldehydes

The first stereoselective, near-equimolar, and metal-free oxidative bioconjugation of amino acids and oligopeptides to aldehydes is presented. Based on a newly developed organocatalytic oxidative concept, the C-terminal and side-chain carboxylic acid functionalities of amino acids and oligopeptides are shown to couple in a stereoselective manner to α-branched aldehydes catalyzed by a chiral primary amine and a quinone as oxidizing agent. The oxidative coupling generally proceeds in high yield. For aspartic acid, selective coupling of the side-chain, or the C-terminal carboxylic acid, is demonstrated depending on the protection strategy. The stereoselective, oxidative bioconjugation concept is extended to a series of oligopeptides where coupling to carboxylic acid functionalities is presented. Bioorthogonal linker molecules for further functionalization are obtained by merging the oxidative coupling strategy with the click concept. It is demonstrated that the configuration of the new stereocenter is determined exclusively by the organocatalyst.