Perfluorinated Iridium Catalyst for Signal Amplification by Reversible Exchange Provides Metal-Free Aqueous Hyperpolarized [1-13C]-Pyruvate.

Hyperpolarized (HP) carbon-13 [13C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-13C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H2) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-13C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-13C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.

Comparative Analysis of Drug-like EP300/CREBBP Acetyltransferase Inhibitors.

The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes.

UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors.

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced in microgram quantities with consistent purity and potency in less than 24 h. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo. The proposed production process is efficient and can be readily scaled up and deployed wherever a viral pathogen might emerge. The current emergence of viral variants of SARS-CoV-2 has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen.

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator.

A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase.

RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small-molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12, that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure-activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12, which has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promoting proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.

An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced with consistent purity and potency in less than 24 hours. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo . The proposed production process is efficient and can be readily scaled up and deployed anywhere in the world where a viral pathogen might emerge. The current emergence of viral variants has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

Structure-Based Optimization of Small Molecule Human Galactokinase Inhibitors.

Classic galactosemia is a rare disease caused by inherited deficiency of galactose-1 phosphate uridylyltransferase (GALT). Accumulation of galactose-1 phosphate (gal-1P) is thought to be the major cause of the chronic complications associated with this disease, which currently has no treatment. Inhibiting galactokinase (GALK1), the enzyme that generates galactose-1 phosphate, has been proposed as a novel strategy for treating classic galactosemia. Our previous work identified a highly selective unique dihydropyrimidine inhibitor against GALK1. With the determination of a co-crystal structure of this inhibitor with human GALK1, we initiated a structure-based structure-activity relationship (SAR) optimization campaign that yielded novel analogs with potent biochemical inhibition (IC50 < 100 nM). Lead compounds were also able to prevent gal-1P accumulation in patient-derived cells at low micromolar concentrations and have pharmacokinetic properties suitable for evaluation in rodent models of galactosemia.

A Universal and High-Throughput Proteomics Sample Preparation Platform.

Major advances have been made to improve the sensitivity of mass analyzers, spectral quality, and speed of data processing enabling more comprehensive proteome discovery and quantitation. While focus has recently begun shifting toward robust proteomics sample preparation efforts, a high-throughput proteomics sample preparation is still lacking. We report the development of a highly automated universal 384-well plate sample preparation platform with high reproducibility and adaptability for extraction of proteins from cells within a culture plate. Digestion efficiency was excellent in comparison to a commercial digest peptide standard with minimal sample loss while improving sample preparation throughput by 20- to 40-fold (the entire process from plated cells to clean peptides is complete in ∼300 min). Analysis of six human cell types, including two primary cell samples, identified and quantified ∼4,000 proteins for each sample in a single high-performance liquid chromatography (HPLC)-tandem mass spectrometry injection with only 100-10K cells, thus demonstrating universality of the platform. The selected protein was further quantified using a developed HPLC-multiple reaction monitoring method for HeLa digests with two heavy labeled internal standard peptides spiked in. Excellent linearity was achieved across different cell numbers indicating a potential for target protein quantitation in clinical research.

High-throughput protein modification quantitation analysis using intact protein MRM and its application on hENGase inhibitor screening.

Proteins are widely used as drug targets, enzyme substrates, and biomarkers for numerous diseases. The emerging demand for proteins quantitation has been increasing in multiple fields. Currently, there is still a big gap for high-throughput protein quantitation at intact protein level using label-free method. Here we choose ribonuclease B (RNB) as a model, which is the substrate for human endo-ß-N-acetylglucosaminidase (hENGase), a promising drug target for the treatment of N-Glycanase deficiency. Intact proteinlevel multiple reaction monitoring (MRM) methods were initally developed and optimized to quantify RNB and deglycosylated RNB (RNB-deg), with the S/N ratio improved by nearly 20-fold compared to the traditional full MS scan methods. To further increase the throughput making it possible for hENGase inhibitors screen, the protein MRM methods were introduced to the RapidFire-MS/MS system, achieving at least 12-fold throughput improvement. This assay was further optimized into 384-well plate format for compound screening with S/B ratio >37-fold and Z' factor >0.7 that is suitable for high-throughput screening of compound collections with a speed of 2 h per 384-well plate and an ability to screen over 3000 compounds per day at a single concentration dose. This 384-well plate based automated SPE-MS/MS assay is efficient and robust for compound screening and the assay format has a wide applicability to protein targets for other disease models.

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

A direct peptide reactivity assay using a high-throughput mass spectrometry screening platform for detection of skin sensitizers.

Chemical-peptide conjugation is the molecular initiating event in skin sensitization. The OECD test guideline uses a high-performance liquid chromatography/ultraviolet (HPLC/UV) detection method to quantify chemical-peptide conjugation in a direct peptide reactivity assay (DPRA), which measures the depletion of two synthetic peptides containing lysine or cysteine residues. To improve assay throughput, sensitivity and accuracy, an automated 384-well plate-based RapidFire solid-phase extraction (SPE) system coupled with tandem mass spectrometry (MS/MS) DPRA was developed and validated in the presence of a newly designed internal standard. Compared to the HPLC/UV-based DPRA, the automated SPE-MS/MS-based DPRA improved throughput from 16 min to 10 s per sample, and substrate peptides usage was reduced from 100 mM to 5 µM. When implementing the SPE-MS/MS-based DPRA into a high-throughput platform, we found 10 compounds that depleted lysine peptide and 24 compounds that depleted cysteine peptide (including 7 unreported chemicals from 55 compounds we tested) in a concentration-response manner. The adduct formation between cysteine and cinnamic aldehyde and ethylene glycol dimethacrylate were further analyzed using high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) to confirm the conjugation. Overall, the automated SPE-MS/MS-based platform is an efficient, economic, and accurate way to detect skin sensitizers.

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

Facile High-Performance Liquid Chromatography Mass Spectrometry Method for Analysis of Cyclocreatine and Phosphocyclocreatine in Complex Mixtures of Amino Acids.

Creatine transporter deficiency (CTD) is caused by a defect in the X-linked creatine transporter SLC6A8 gene leading to severe neurologic and physiologic conditions. Cyclocreatine and phosphocyclocreatine supplementation is seen as a potential treatment, but the presence of these compounds within commercially available dietary supplements presents the risk of self-medication. High-performance liquid chromatography-mass spectrometry (HPLC-MS) is an excellent technique to assess composition of complex amino acid mixtures. Herein, we have developed a facile HPLC-MS method using a cyano column in hydrophilic interaction liquid chromatography (HILIC) mode with isocratic elution over 4 min to identify the main components of two commercially available dietary supplements. The relative standard deviation (RSD) for retention time and extracted ion integrated area are <0.3% and 4%, respectively, showing excellent reproducibility. Cyclocreatine and phosphocyclocreatine were not detectable within the dietary supplements, even at ppm levels, demonstrating the power and importance of the developed HPLC-MS method in analyzing complex mixtures.

High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.

A novel class of ion displacement ligands as antagonists of the αIIbß3 receptor that limit conformational reorganization of the receptor.

A collection of αIIbß3 integrin receptor antagonists possessing a unique MIDAS metal ion displacement mechanism of action is presented. Insight into these agents' structure-activity relationships, binding modality, and pharmacokinetic and pharmacodynamic profiles highlight the potential of these small molecule ion displacement ligands as attractive candidates for clinical development.

Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A.

The racemic product of the Betti reaction of 5-chloro-8-hydroxyquinoline, benzaldehyde and 2-aminopyridine was separated by chiral HPLC to determine which enantiomer inhibited botulinum neurotoxin serotype A. When the enantiomers unexpectedly proved to have comparable activity, the absolute structures of (+)-(R)-1 and (-)-(S)-1 were determined by comparison of calculated and observed circular dichroism spectra. Molecular modeling studies were undertaken in an effort to understand the observed bioactivity and revealed different ensembles of binding modes, with roughly equal binding energies, for the two enantiomers.

Phosphodiesterase 4 inhibitors enhance sexual pleasure-seeking activity in rodents.

Pleasure-seeking deficits, including lack of libido, are a core feature of depression. Animal and preliminary clinical studies both suggest that phosphodiesterase 4 (PDE4) is a target for developing novel antidepressants. This study examined the potential involvement of PDE4 in the pathology of depression in both animal models and human postmortem brains. In humans, PDE4B and PDE4D levels were elevated in cingulate cortical tissue from individuals with major depressive disorder (MDD) compared to controls. Using the female urine smelling test (FUST), a recently refined method for monitoring sexual pleasure-seeking activity in mice, we found that icv infusion of selective potent PDE4 inhibitors enhanced sexual pleasure-seeking activity in male mice that underwent the learned helplessness or serotonin depletion paradigms. The infusion also increased sexual pleasure-seeking activity in naïve male mice. The results suggest that PDE4 may be a plausible contributor to the sexual pleasure-seeking deficits seen in depressed patients; inhibiting PDE4 may restore these deficits.

Total synthesis of LL-Z1640-2 utilizing a late-stage intramolecular Nozaki-Hiyama-Kishi reaction.

A total synthesis of LL-Z1640-2 (2), a potent and selective kinase inhibitor, has been completed. The key step of the convergent synthesis utilized a late-stage intramolecular Nozaki-Hiyama-Kishi (NHK) reaction to close the macrocycle at the C6'-C7' bond.