Jennifer E. Golden

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe

Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases. Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays. Conclusion: The identified compound is a highly Cdc42-selective inhibitor. Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research. Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

Syntheses of the Stemona Alkaloids (±)-Stenine, (±)-Neostenine, and (±)-13-Epineostenine Using a Stereodivergent Diels-Alder/Azido-Schmidt Reaction

A tandem Diels-Alder/azido-Schmidt reaction sequence provides rapid access to the core skeleton shared by several Stemona alkaloids including stenine, neostenine, tuberostemonine, and neotuberostemonine. The discovery and evolution of inter- and intramolecular variations of this process and their applications to total syntheses of (+/-)-stenine and (+/-)-neostenine are described. The stereochemical outcome of the reaction depends on both substrate type and reaction conditions, enabling the preparation of both (+/-)-stenine and (+/-)-neostenine from the same diene/dienophile combination.

A Combined Intramolecular Diels–Alder/Intramolecular Schmidt Reaction: Formal Synthesis of (±)‐Stenine

Financial support was provided by the National Institutes of Health (Grant GM-49093). J. Golden gratefully acknowledges the Madison A. and Lila Self Fellowship Program for its support. The authors also thank Dr. David Vander Velde for NMR assistance and Dr. Doug Powell for crystallographic data.

A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition.

Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.

Discovery of AMG 369, a Thiazolo[5,4-b]pyridine Agonist of S1P1 and S1P5.

The optimization of a series of thiazolopyridine S1P1 agonists with limited activity at the S1P3 receptor is reported. These efforts resulted in the discovery of 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]pyridin-2-yl)benzyl)azetidine-3-carboxylic acid (5d, AMG 369), a potent dual S1P1/S1P5 agonist with limited activity at S1P3 and no activity at S1P2/S1P4. Dosed orally at 0.1 mg/kg, 5d is shown to reduce blood lymphocyte counts 24 h postdose and delay the onset and reduce the severity of experimental autoimmune encephalomyelitis in rat.

Evaluation of anti-Zika virus activities of broad-spectrum antivirals and NIH clinical collection compounds using a cell-based, high-throughput screen assay

Abstract Recent studies have clearly underscored the association between Zika virus (ZIKV) and severe neurological diseases such as microcephaly and Guillain‐Barre syndrome. Given the historical complacency surrounding this virus, however, no significant antiviral screenings have been performed to specifically target ZIKV. As a result, there is an urgent need for a validated screening method and strategy that is focused on highlighting potential anti‐ZIKV inhibitors that can be further advanced via rigorous validation and optimization. To address this critical gap, we sought to test whether a cell‐based assay that measures protection from the ZIKV‐induced cytopathic effect could serve as a high‐throughput screen assay for discovering novel anti‐ZIKV inhibitors. Employing this approach, we tested the anti‐ZIKV activity of previously known broad‐spectrum antiviral compounds and discovered several compounds (e.g., NITD008, SaliPhe, and CID 91632869) with anti‐ZIKV activity. Interestingly, while GTP synthesis inhibitors (e.g., ribavirin or mycophenolic acid) were too toxic or showed no anti‐ZIKV activity (EC50 > 50 &mgr;M), ZIKV was highly susceptible to pyrimidine synthesis inhibitors (e.g., brequinar) in the assay. We amended the assay into a high‐throughput screen (HTS)‐compatible 384‐well format and then screened the NIH Clinical Compound Collection library, which includes a total of 727 compounds organized, using an 8‐point dose response format with two Zika virus strains (MR766 and PRVABC59, a recent human isolate). The screen discovered 6‐azauridine and finasteride as potential anti‐ZIKV inhibitors with EC50 levels of 3.18 and 9.85 &mgr;M for MR766, respectively. We further characterized the anti‐ZIKV activity of 6‐azauridine and several pyrimidine synthesis inhibitors such as brequinar in various secondary assays including an antiviral spectrum test within flaviviruses and alphaviruses, Western blot (protein), real‐time PCR (RNA), and plaque reduction assays (progeny virus). From these assays, we discovered that brequinar has potent anti‐ZIKV activity. Our results show that a broad anti‐ZIKV screen of compound libraries with our CPE‐based HTS assay will reveal multiple chemotypes that could be pursued as lead compounds for therapies to treat ZIKV‐associated diseases or as molecular probes to study the biology of the ZIKV replication mechanism. HighlightsWe developed a CPE‐based assay for Zika virus inhibitor screen.Zika virus is susceptible to several broad‐spectrum antivirals including SaliPhe, CID 91632869, and NITD008.We screened the NIH Clinical Compound Collection, 727 compounds, using a dose response format with two Zika virus strains.The screen discovered 6‐azauridine and finasteride as potential anti‐ZIKV inhibitors.Zika virus shows the highest sensitivity to brequinar among pyrimidine synthesis inhibitors.

Structure-guided design of substituted aza-benzimidazoles as potent hypoxia inducible factor-1α prolyl hydroxylase-2 inhibitors

We report the structure-based design and synthesis of a novel series of aza-benzimidazoles as PHD2 inhibitors. These efforts resulted in compound 22, which displayed highly potent inhibition of PHD2 function in vitro.

Interrogating a Hexokinase-Selected Small-Molecule Library for Inhibitors of Plasmodium falciparum Hexokinase

ABSTRACT Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (∼26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 μM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥0.6 and <6.8 μM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

Potent and Selective Inhibitors of the TASK-1 Potassium Channel Through Chemical Optimization of a Bis-amide Scaffold

TASK-1 is a two-pore domain potassium channel that is important to modulating cell excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for therapeutic benefit, its physiological role needs better characterization; however, designing selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1 selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective hit led to analog 35 which displays a TASK-1 IC50=16 nM with 62-fold selectivity over TASK-3 in an orthogonal electrophysiology assay.