Small-molecule targeting of GPCR-independent noncanonical G-protein signaling in cancer.

Activation of heterotrimeric G-proteins (Gαßγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class small-molecule inhibitor of noncanonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking noncanonical G-protein signaling in tumor cells and inhibiting proinvasive traits of metastatic cancer cells. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable noncanonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.

Predicting Emission Wavelengths in Benzobisoxazole-Based OLEDs with Gradient Boosted Ensemble Models.

We demonstrate the use of gradient-boosted ensemble models that accurately predict emission wavelengths in benzobis[1,2-d:4,5-d']oxazole (BBO) based fluorescent emitters. We have curated a database of 50 molecules from previously published data by the Jeffries-EL group using density functional theory (DFT) computed ground and excited state features. We consider two machine learning (ML) models based on (i) whole cruciform molecules and (ii) their constituent fragment molecules. Both ML models provide accurate predictions with root-mean-square errors between 30 and 36 nm, competitive with state-of-the-art deep learning models trained on orders of magnitude more molecules, and this accuracy holds even when tested on four new BBO emitters unseen by the models. We also provide an interpretable feature importance analysis and discuss the relevant relationships between DFT and changes in predicted emission wavelength.

Synthesis of Neocannabinoids Using Controlled Friedel-Crafts Reactions.

A one-step transformation to produce 8,9-dihydrocannabidiol (H2CBD) and related "neocannabinoids" via controlled Friedel-Crafts reactions is reported. Experimental and computational studies probing the mechanism of neocannabinoid synthesis from cyclic allylic alcohol and substituted resorcinol reaction partners provide understanding of the kinetic and thermodynamic factors driving regioselectivity for the reaction. Herein, we present the reaction scope for neocannabinoid synthesis including the production of both normal and abnormal isomers under both kinetic and thermodynamic control. Discovery and optimization of this one-step protocol between various allylic alcohols and resorcinol derivatives are discussed and supported with density functional theory calculations.

Mechanisms of nucleophosmin (NPM)-mediated regulated cell death elucidated by Hsp70 during renal ischemia.

Nucleophosmin (NPM), a nucleolar-based protein chaperone, promotes Bax-mediated mitochondrial injury and regulates cell death during acute kidney injury. However, the steps that transform NPM from an essential to a toxic protein during stress are unknown. To localize NPM-mediated events causing regulated cell death during ischemia, wild type (WT) and Hsp70 mutant proteins with characterized intracellular trafficking defects that restrict movement to either the nucleolar region (M45) or cytosol (985A) were expressed in primary murine proximal tubule epithelial cells (PTEC) harvested from Hsp70 null mice. After ischemia in vitro, PTEC survival was significantly improved and apoptosis reduced in rank order by selectively overexpressing WT > M45 > 985A Hsp70 proteins. Only Hsp70 with nuclear access (WT and M45) inhibited T95 NPM phosphorylation responsible for NPM translocation and also reduced cytosolic NPM accumulation. In contrast, WT or 985A > M45 significantly improved survival in Hsp70 null PTEC that expressed a cytosol-restricted NPM mutant, more effectively bound NPM, and also reduced NPM-Bax complex formation required for mitochondrial injury and cell death. Hsp70 knockout prevented the cytoprotective effect of suppressing NPM in ischemic PTEC and also increased cytosolic NPM accumulation after acute renal ischemia in vivo, emphasizing the inhibitory effect of Hsp70 on NPM-mediated toxicity. Distinct cytoprotective mechanisms by wild type and mutant Hsp70 proteins identify dual nuclear and cytosolic events that mediate NPM toxicity during stress-induced apoptosis and are rational targets for therapeutic AKI interventions. Antagonizing these early events in regulated cell death promotes renal cell survival during experimental AKI.

Recapitulating the Binding Affinity of Nrf2 for KEAP1 in a Cyclic Heptapeptide, Guided by NMR, X-ray Crystallography, and Machine Learning.

Macrocycles, including macrocyclic peptides, have shown promise for targeting challenging protein-protein interactions (PPIs). One PPI of high interest is between Kelch-like ECH-Associated Protein-1 (KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided by X-ray crystallography, NMR, modeling, and machine learning, we show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be recapitulated in a cyclic 7-mer peptide, c[(D)-ß-homoAla-DPETGE]. This compound was identified from the Nrf2-derived linear peptide GDEETGE (KD = 4.3 µM) solely by optimizing the conformation of the cyclic compound, without changing any KEAP1 interacting residue. X-ray crystal structures were determined for each linear and cyclic peptide variant bound to KEAP1. Despite large variations in affinity, no obvious differences in the conformation of the peptide binding residues or in the interactions they made with KEAP1 were observed. However, analysis of the X-ray structures by machine learning showed that locations of strain in the bound ligand could be identified through patterns of subangstrom distortions from the geometry observed for unstrained linear peptides. We show that optimizing the cyclic peptide affinity was driven partly through conformational preorganization associated with a proline substitution at position 78 and with the geometry of the noninteracting residue Asp77 and partly by decreasing strain in the ETGE motif itself. This approach may have utility in dissecting the trade-off between conformational preorganization and strain in other ligand-receptor systems. We also identify a pair of conserved hydrophobic residues flanking the core DxETGE motif which play a conformational role in facilitating the high-affinity binding of Nrf2 to KEAP1.

Reversible PCET and Ambient Catalytic Oxidative Alcohol Dehydrogenation by {V=O} Perfluoropinacolate Complexes.

A new air-stable catalyst for the oxidative dehydrogenation of benzylic alcohols under ambient conditions has been developed. The synthesis and characterization of this compound and the related monomeric and dimeric V(IV)- and V(V)-pinF (pinF = perfluoropinacolate) complexes are reported herein. Monomeric V(IV) complex (Me4N)2[V(O)(pinF)2] (1) and dimeric (µ-O)2-bridged V(V) complex (Me4N)2[V2(O)2(µ-O)2(pinF)2] (3a) are prepared in water under ambient conditions. Monomeric V(V) complex (Me4N)[V(O)(pinF)2] (2) may be generated via chemical oxidation of 1 under an inert atmosphere, but dimerizes to 3a upon exposure to air. Complexes 1 and 2 display a perfectly reversible VIV/V couple at 20 mV (vs Ag/AgNO3), whereas a quasi-reversible VIV/V couple at -865 mV is found for 3a. Stoichiometric reactions of 3a with both fluorenol and TEMPOH result in the formation of (Me4N)2[V2(O)2(µ-OH)2(pinF)2] (4a), which contains two V(IV) centers that display antiferromagnetic coupling. In order to structurally characterize the dinuclear anion of 4a, {K(18C6)}+ countercations were employed, which formed stabilizing K···O interactions between the counterion and each terminal oxo moiety and H-bonding between the oxygen atoms of the crown ether and µ-OH bridges of the dimer, resulting in {K(18C6)}2[V2(O)2(µ-OH)2(pinF)2] (4b). The formal storage of H2 in 4a is reversible and proton-coupled electron transfer (PCET) from crystals of 4a regenerates 3a upon exposure to air over the course of several days. Furthermore, the reaction of 3a (2%) under ambient conditions with excess fluorenol, cinnamyl alcohol, or benzyl alcohol resulted in the selective formation of fluorenone (82% conversion), cinnamaldehyde (40%), or benzaldehyde (7%), respectively, reproducing oxidative alcohol dehydrogenation (OAD) chemistry known for VOx surfaces and demonstrating, in air, the thermodynamically challenging selective oxidation of alcohols to aldehydes/ketones.

One-pot synthesis of epoxides from benzyl alcohols and aldehydes.

A one-pot synthesis of epoxides from commercially available benzyl alcohols and aldehydes is described. The reaction proceeds through in situ generation of sulfonium salts from benzyl alcohols and their subsequent deprotonation for use in Corey-Chaykovsky epoxidation of aldehydes. The generality of the method is exemplified by the synthesis of 34 epoxides that were made from an array of electronically and sterically varied alcohols and aldehydes.

Identification of Anti-prion Compounds using a Novel Cellular Assay.

Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.

A photochemical flow reactor for large scale syntheses of aglain and rocaglate natural product analogues.

Herein, we report the development of continuous flow photoreactors for large scale ESIPT-mediated [3+2]-photocycloaddition of 2-(p-methoxyphenyl)-3-hydroxyflavone and cinnamate-derived dipolarophiles. These reactors can be efficiently numbered up to increase throughput two orders of magnitude greater than the corresponding batch reactions.

[2+2] Photocycloaddition of Cinnamates in Flow and Development of a Thiourea Catalyst.

Cyclobutanes derived from the dimerization of cinnamic acids are the core scaffolds of many molecules with potentially interesting biological activities. By utilizing a powerful flow photochemistry platform developed in our laboratory, we have evaluated the effects of flow on the dimerization of a range of cinnamate substrates. During the course of the study we also identified a bis(thiourea) catalyst that facilitates better reactivity and moderate diastereoselectivity in the reaction. Overall, we show that carrying out the reaction in flow in the presence of the catalyst affords consistent formation of predictable cyclobutane diastereomers.

Multidimensional reaction screening for photochemical transformations as a tool for discovering new chemotypes.

We have developed an automated photochemical microfluidics platform that integrates a 1 kW high-pressure Hg vapor lamp and allows for analytical pulse flow or preparative continuous flow reactions. Herein, we will discuss the use of this platform toward the discovery of new chemotypes through multidimensional reaction screening. We will highlight the ability to discretely control wavelengths with optical filters, allowing for control of reaction outcomes.

Discovery of new antimalarial chemotypes through chemical methodology and library development.

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

Accessing Cyclobutane Polymers: Overcoming Synthetic Challenges via Efficient Continuous Flow [2 + 2] Photopolymerization.

We report an improved and efficient method to prepare well-defined, structurally complex truxinate cyclobutane polymers via a thioxanthone sensitized solution state [2 + 2] photopolymerization. Monomers with varying electron density and structure polymerize in good to excellent yields to afford a library of 42 polyesters. Monomers with internal olefin separation distances of greater than 5 Å undergo polymerization via intermolecular [2 + 2] photocycloaddition readily, as opposed to the intramolecular [2 + 2] photocycloaddition observed in monomers with olefins in closer proximity. Use of a continuous flow reactor decreases reaction time, increases polymer molecular weight, and decreases dispersity compared to batch reactions. Furthermore, under continuous flow, polymerization is readily scalable beyond what is possible with batch reactions. This advancement ushers truxinate cyclobutane-based polyesters, which have been historically limited to a few examples and only research scale quantities, to the forefront of development as new materials for potential use across industry sectors.

Sigma Receptor Ligands Are Potent Antiprion Compounds that Act Independently of Sigma Receptor Binding.

Prion diseases are invariably fatal neurodegenerative diseases of humans and other animals for which there are no effective treatment options. Previous work from our laboratory identified phenethylpiperidines as a novel class of anti-prion compounds. While working to identify the molecular target(s) of these molecules, we unexpectedly discovered ten novel antiprion compounds based on their known ability to bind to the sigma receptors, σ1R and σ2R, which are currently being tested as therapeutic or diagnostic targets for cancer and neuropsychiatric disorders. Surprisingly, however, knockout of the respective genes encoding σ1R and σ2R (Sigmar1 and Tmem97) in prion-infected N2a cells did not alter the antiprion activity of these compounds, demonstrating that these receptors are not the direct targets responsible for the antiprion effects of their ligands. Further investigation of the most potent molecules established that they are efficacious against multiple prion strains and protect against downstream prion-mediated synaptotoxicity. While the precise details of the mechanism of action of these molecules remain to be determined, the present work forms the basis for further investigation of these compounds in preclinical studies. Given the therapeutic utility of several of the tested compounds, including rimcazole and haloperidol for neuropsychiatric conditions, (+)-pentazocine for neuropathic pain, and the ongoing clinical trials of SA 4503 and ANAVEX2-73 for ischemic stroke and Alzheimer's disease, respectively, this work has immediate implications for the treatment of human prion disease.

High-throughput infrared spectroscopy for quantification of peptides in drug discovery.

Peptides have gained an increasing importance in drug discovery as potential therapeutics. Discovery efforts toward finding new, efficacious peptide-based therapeutics have increased the throughput of peptide development, allowing the rapid generation of unique and pure peptide samples. However, high-throughput analysis of peptides may be still challenging and can encumber a high-throughput drug discovery campaign. We report herein a fit-for-purpose method to quantify peptide concentrations using high-throughput infrared spectroscopy (HT-IR). Through the development of this method, multiple critical method parameters were optimized including solvent composition, droplet deposition size, plate drying procedures, sample concentration, and internal standard. The relative absorbance of the amide region (1600-1750 cm-1) to the internal standard, K3Fe(CN)6 (2140 cm-1), was determined to be most effective at providing lowest interference for measuring peptide concentration. The best sample deposition was achieved by dissolving samples in a 50:50 v/v allyl alcohol/water mixture. The developed method was used on 96-well plates and analyzed at a rate of 22 min per plate. Calibration curves to measure sample concentration versus response relationship displayed sufficient linearity (R2 > 0.95). The repeatability and scope of detection was demonstrated with eighteen peptide samples that were measured with most values below 20% relative standard deviation. The linear dynamic range of the method was determined to be between 1 and 5 mg/mL. This developed HT-IR methodology could be a useful tool in peptide drug candidate lead identification and optimization processes.

Sigma receptor ligands are potent anti-prion compounds that act independently of sigma receptor binding.

Prion diseases are invariably fatal neurodegenerative diseases of humans and other animals for which there are no treatment options. Previous work from our laboratory identified phenethyl piperidines as novel class of anti-prion compounds. While working to identify the molecular target(s) of these molecules, we unexpectedly discovered ten novel anti-prion compounds based on their known ability to bind to the sigma receptors, σ 1 R and 2 R, which are currently being tested as therapeutic or diagnostic targets for cancer and neuropsychiatric disorders. Surprisingly, however, knockout of the respective genes encoding σ 1 R and σ 2 R ( Sigmar1 and Tmem97 ), in prion infected N2a cells did not alter the anti-prion activity of these compounds, demonstrating that these receptors are not the direct targets responsible the anti-prion effects of their ligands. Further investigation of the most potent molecules established that they are efficacious against multiple prion strains and protect against downstream prion-mediated synaptotoxicity. While the precise details of the mechanism of action of these molecules remains to be determined, the present work forms the basis for further investigations of these compounds in pre-clinical studies. Given the therapeutic utility of several of the tested compounds, including rimcazole and haloperidol for neuropsychiatric conditions, (+)-pentazocine for neuropathic pain, and the ongoing clinical trials of SA 4503 and ANAVEX2-73 for ischemic stroke and Alzheimer's disease, respectively, this work has immediate implications for the treatment of human prion disease.

Small-molecule targeting of GPCR-independent non-canonical G protein signaling inhibits cancer progression.

Activation of heterotrimeric G-proteins (Gαßγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically-approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class smallmolecule inhibitor of non-canonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking non-canonical G-protein signaling in tumor cells, and inhibiting pro-invasive traits of metastatic cancer cells in vitro and in mice. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable non-canonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.

Synthesis of a novel chemotype via sequential metal-catalyzed cycloisomerizations.

Sequential cycloisomerizations of diynyl o-benzaldehyde substrates to access novel polycyclic cyclopropanes are reported. The reaction sequence involves initial Cu(I)-mediated cycloisomerization/nucleophilic addition to an isochromene followed by diastereoselective Pt(II)-catalyzed enyne cycloisomerization.

Synthesis and reactivity of bicyclo[3.2.1]octanoid-derived cyclopropanes.

Photochemical oxa-di-π-methane rearrangement of bicyclo[3.2.1]octanoid scaffolds affords multifunctional, donor-acceptor cyclopropanes. A related photochemical reaction of an iminium ether substrate uncovered an unprecedented aza-di-π-methane rearrangement of a ß,γ-unsaturated iminium. Donor-acceptor cyclopropanes have been evaluated as substrates for reactions generating several new chemotypes.