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 Glucokinases from Pathogenic Free-Living Amoebae.

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

Dihydropyrazinoquinazolinones via SN2 Sulfamidate Ring-Opening and a Sequential Quinazolinone-Amidine Rearrangement Strategy (SQuAReS).

A synthesis of dihydropyrazino-[2,1-b]-quinazolinones is described using a 2-alkylaminoquinazolinone-mediated ring opening of a-/chiral sulfamidates, followed by a tandem quinazolinone-amidine rearrangement termed SQuAReS. This approach takes advantage of sulfamidates whose regioselective ring opening, after hydrolysis, appends an optimally distanced nucleophilic amine to a quinazolinone such that subsequent domino rearrangements are favored, integrating unique substitution patterns on a privileged core. This three-step protocol integrated five telescoped transformations and generated 20 pyrazinoquinazolinones in up to 74% yield with high enantiomeric fidelity and diastereoselectivity.

Emergence and Magnitude of ML336 Resistance in Venezuelan Equine Encephalitis Virus Depend on the Microenvironment.

Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that can cause neurological disease and death in humans and equines following transmission from infected mosquitoes. Despite the continued epidemic threat of VEEV, and its potential use as a bioterrorism agent, there are no FDA-approved antivirals or vaccines for treatment or prevention. Previously, we reported the discovery of a small molecule, ML336, with potent antiviral activity against VEEV. To further explore the population-level resistance profiles of ML336, we developed a whole-genome next-generation sequencing (NGS) approach to examine single nucleotide polymorphisms (SNPs) from virus passaged in dose escalation studies in a nonhuman primate kidney epithelial and a human astrocyte cell line, Vero 76 and SVGA, respectively. We passaged VEEV TC-83 in these two cell lines over seven concentrations of ML336, starting at 50 nM. NGS revealed several prominent mutations in the nonstructural protein (nsP) 3 and nsP4 genes that emerged consistently in these two distinct in vitro environments-notably, a mutation at Q210 in nsP4. Several of these mutations were stable following passaging in the absence of ML336 in Vero 76 cells. Network analyses showed that the trajectory of resistance differed between Vero and SVGA. Moreover, the penetration of SNPs was lower in SVGA. In conclusion, we show that the microenvironment influenced the SNP profile of VEEV TC-83. Understanding the dynamics of resistance in VEEV against newly developed antiviral compounds will guide the design of optimal drug candidates and dosing regimens for minimizing the emergence of resistant viruses.IMPORTANCE RNA viruses, including Venezuelan equine encephalitis virus (VEEV), have high mutation rates that allow for rapid adaptation to selective pressures in their environment. Antiviral compounds exert one such pressure on virus populations during infections. Next-generation sequencing allows for examination of viruses at the population level, which enables tracking of low levels of single-nucleotide polymorphisms in the population over time. Therefore, the timing and extent of the emergence of resistance to antivirals can be tracked and assessed. We show here that in VEEV, the trajectory and penetration of antiviral resistance reflected the microenvironment in which the virus population replicates. In summary, we show the diversity of VEEV within a single population under antiviral pressure and two distinct cell types, and we show that population dynamics in these viruses can be examined to better understand how they evolve over time.

Construction of N-Boc-2-Alkylaminoquinazolin-4(3H)-Ones via a Three-Component, One-Pot Protocol Mediated by Copper(II) Chloride that Spares Enantiomeric Purity.

Chiral 2-alkylquinazolinones are key synthetic intermediates, but their preparation in high optical purity is challenging. Thus, a multicomponent procedure integrating anthranilic acids, N-Boc-amino acids, and amines in the presence of methanesulfonyl chloride, N-methylimidazole, and copper(II) chloride was developed to mildly afford N-Boc-2-alkylaminoquinazolin-4(3H)-ones with excellent preservation of enantiomeric purity (>99% ee). Copper(II) chloride was essential to retaining enantiopurity, and reaction component structural changes were well tolerated, resulting in an efficient, all-in-one procedure that promotes sequential coupling, lactonization, aminolysis, and cyclization in good yields. The method was applied to the rapid assembly of four key intermediates used in the synthesis of high profile quinazolinones, including several PI3K inhibitor drugs.

Divergent 2-Chloroquinazolin-4(3H)-one Rearrangement: Twisted-Cyclic Guanidine Formation or Ring-Fused N-Acylguanidines via a Domino Process.

A highly efficient 2-chloroquinazolin-4(3H)-one rearrangement was developed that predictably generates either twisted-cyclic or ring-fused guanidines in a single operation, depending on the presence of a primary versus secondary amine in the accompanying diamine reagent. Exclusive formation of twisted-cyclic guanidines results from pairing 2-chloroquinazolinones with secondary diamines. Use of primary amine-containing diamines permits a domino quinazolinone rearrangement/intramolecular cyclization, gated through (E)-twisted-cyclic guanidines, to afford ring-fused N-acylguanidines. This scalable, structurally tolerant transformation generated 55 guanidines and delivered twisted-cyclic guanidines with robust plasma stability and an abbreviated total synthesis of an antitumor ring-fused guanidine (4 steps, 55 % yield).

Synthesis of Ring-Fused, N-Substituted 4-Quinolinones Using pKa-Guided, Base-Promoted Annulations with Isatoic Anhydrides: Total Synthesis of Penicinotam.

An anionic annulation strategy employing isatoic anhydrides and a wide assortment of enolizable partners was developed to afford over 80 novel ring-fused, N-substituted 4-quinolinones, an underrepresented privileged template. Multiple factors governing the efficiency of the transformation were determined, resulting in a reliable and tunable synthetic platform applicable for a broad range of substrates with variable deprotonation susceptibility, such as tetramic and tetronic acids, cyclic 1,3-diketones, and cycloalkanones. Application to the synthesis of bioactive, pyrrolizine-fused 4-quinolinone, penicinotam 3, resulted in the most brief and highest yielding total synthesis of the alkaloid in three steps and a 36% overall yield.

Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase.

Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (∼98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The first enzyme required for glucose metabolism is typically a hexokinase (HK), which transfers a phosphate from ATP to glucose. The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. However, the N. fowleri genome lacks an obvious HK homolog and instead harbors a glucokinase (Glck). The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (Homo sapiens Glck), suggesting that parasite-specific inhibitors with anti-amoeba activity can be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with the greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 ± 7.3 µM and 141.6 ± 9.9 µM for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2-Å resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose-phosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of <1 µM that may prove useful as hit chemotypes for further leads and therapeutic development against N. fowleri.

Telescoped synthesis of C3-functionalized (E)-arylamidines using Ugi-Mumm and regiospecific quinazolinone rearrangements.

An efficient four-step, six-transformation protocol was developed to afford bioactive N-alkyl- or N-arylamide (E)-arylamidines featuring strategic amidine C3 modifications which were inaccessible or low yielding by previous methods. This synthetic approach, exemplified with 24 amidines and requiring only a single purification, highlights a multicomponent Ugi-Mumm rearrangement to afford highly diversified quinazolinones which undergo regiospecific rearrangement to afford new amidines. The method extensively broadens the structural scope of this new class of trisubstituted amidines and demonstrates the tolerance of regional C3 amidine steric bulk, visualized with X-ray crystallographic analysis.

Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells.

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

Identification and Characterization of Influenza Virus Entry Inhibitors through Dual Myxovirus High-Throughput Screening.

UNLABELLED: Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCE: This study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.

Antiparasitic lethality of sulfonamidebenzamides in kinetoplastids.

A sulfonamidebenzamide series was assessed for anti-kinetoplastid parasite activity based on structural similarity to the antiparasitic drug, nifurtimox. Through structure-activity optimization, derivatives with limited mammalian cell toxicity and increased potency toward African trypanosomes and Leishmania promastigotes were developed. Compound 22 had the best potency against the trypanosome (EC50=0.010µM) while several compounds showed ∼10-fold less potency against Leishmania promastigotes without impacting mammalian cells (EC50>25µM). While the chemotype originated from an unrelated optimization program aimed at selectively activating an apoptotic pathway in mammalian cancer cells, our preliminary results suggest that a distinct mechanism of action from that observed in mammalian cells is responsible for the promising activity observed in parasites.

Evaluation of substituted ebselen derivatives as potential trypanocidal agents.

Human African trypanosomiasis is a disease of sub-Saharan Africa, where millions are at risk for the illness. The disease, commonly referred to as African sleeping sickness, is caused by an infection by the eukaryotic pathogen, Trypanosoma brucei. Previously, a target-based high throughput screen revealed ebselen (EbSe), and its sulfur analog, EbS, to be potent in vitro inhibitors of the T. brucei hexokinase 1 (TbHK1). These molecules also exhibited potent trypanocidal activity in vivo. In this manuscript, we synthesized a series of sixteen EbSe and EbS derivatives bearing electron-withdrawing carboxylic acid and methyl ester functional groups, and evaluated the influence of these substituents on the biological efficacy of the parent scaffold. With the exception of one methyl ester derivative, these modifications ablated or blunted the potent TbHK1 inhibition of the parent scaffold. Nonetheless, a few of the methyl ester derivatives still exhibited trypanocidal effects with single-digit micromolar or high nanomolar EC50 values.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 µM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.

Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State.

Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

One-pot, regiospecific assembly of (E)-benzamidines from δ- and γ-amino acids via an intramolecular aminoquinazolinone rearrangement.

The efficient generation of novel, N-linked benzamidines resulting from a regiospecific rearrangement of quinazolinones is described. This methodology study explored reaction parameters including the effect of changing solvent and temperature, as well as varying electronic substituents on the structural core. The transformation was extensively optimized in terms of reaction conditions and scope, resulting in a protocol that consistently affords diversely functionalized amidines in high yield. The process permits regional structural derivatization that was previously inaccessible, and the multistep process was also reduced to a telescoped, five-step sequence that efficiently affords pharmacologically unique (E)-benzamidoamidines from N-BOC protected γ- and δ-amino acids.

Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.

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.

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.

Pharmacophore Establishment and Optimization of Saturated 1,6-Naphthyridine-Fused Quinazolinones that Inhibit Meningoencephalitis-Causing Naegleria fowleri.

Primary amoebic meningoencephalitis (PAM) is a human brain infection caused by Naegleria fowleri with a 97% mortality rate. Quinazolinones resulting from a Mannich-coupled domino rearrangement were recently identified as inhibitors of the amoeba. Herein, we resolved the effective concentrations for 25 pilot compounds and then, using the Mannich protocol and a key late-stage, N-demethylation/functionalization, we synthesized 53 additional analogs to improve potency, solubility and microsomal stability. We established an antiamoebic quinazolinone pharmacophore, culminating in (±)-trans-57b which featured the best combination of potency, selectivity index, solubility, and microsomal stability. Enantiomeric separation afforded (4aS,13bR)-57b (BDGR-20237) with a 41-fold potency advantage over its enantiomer. ADME and mouse pharmacokinetic profiling for BDGR-20237 revealed high brain penetrance but a limited half-life which did not statistically enhance the mouse survival in a pilot efficacy study. The pharmacophoric model, supported by 88 quinazolinones, several of which exhibit subnanomolar potency, will guide further scaffold optimization.