Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma.

Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F0F1 ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.

Novel orexin receptor agonists based on arene- or pyridine-fused 1,3-dihydro-2H-imidazole-2-imines.

A structurally novel class of benzo- or pyrido-fused 1,3-dihydro-2H-imidazole-2-imines was designed and evaluated in an inositol phosphate accumulation assay for Gq signaling to measure agonistic activation of the orexin receptor type 2 (OX2R). These compounds were synthesized in 4-9 steps overall from readily available starting materials. Analogs that contain a stereogenic methyl or cyclopropyl substituent at the benzylic center, and a correctly configured alkyl ether, alkoxyalkyl ether, cyanoalkyl ether, or α-hydroxyacetamido substituted homobenzylic sidechain were identified as the most potent activators of OX2R coupled Gq signaling. Our results also indicate that agonistic activity was stereospecific at both the benzylic and homobenzylic stereogenic centra. We identified methoxyethoxy-substituted pyrido-fused dihydroimidazolimine analog 63c containing a stereogenic benzylic methyl group was the most potent agonist, registering a respectable EC50 of 339 nM and a maximal response (Emax) of 96 % in this assay. In vivo pharmacokinetic analysis indicated good brain exposure for several analogs. Our combined results provide important information towards a structurally novel class of orexin receptor agonists distinct from current chemotypes.

Evaluation of the efficacy of the hypocretin/orexin receptor agonists TAK-925 and ARN-776 in narcoleptic orexin/tTA; TetO-DTA mice.

The sleep disorder narcolepsy, a hypocretin deficiency disorder thought to be due to degeneration of hypothalamic hypocretin/orexin neurons, is currently treated symptomatically. We evaluated the efficacy of two small molecule hypocretin/orexin receptor-2 (HCRTR2) agonists in narcoleptic male orexin/tTA; TetO-DTA mice. TAK-925 (1-10 mg/kg, s.c.) and ARN-776 (1-10 mg/kg, i.p.) were injected 15 min before dark onset in a repeated measures design. EEG, EMG, subcutaneous temperature (Tsc ) and activity were recorded by telemetry; recordings for the first 6 h of the dark period were scored for sleep/wake and cataplexy. At all doses tested, TAK-925 and ARN-776 caused continuous wakefulness and eliminated sleep for the first hour. Both TAK-925 and ARN-776 caused dose-related delays in NREM sleep onset. All doses of TAK-925 and all but the lowest dose of ARN-776 eliminated cataplexy during the first hour after treatment; the anti-cataplectic effect of TAK-925 persisted into the second hour for the highest dose. TAK-925 and ARN-776 also reduced the cumulative amount of cataplexy during the 6 h post-dosing period. The acute increase in wakefulness produced by both HCRTR2 agonists was characterised by increased spectral power in the gamma EEG band. Although neither compound provoked a NREM sleep rebound, both compounds affected NREM EEG during the second hour post-dosing. TAK-925 and ARN-776 also increased gross motor activity, running wheel activity, and Tsc , suggesting that the wake-promoting and sleep-suppressing activities of these compounds could be a consequence of hyperactivity. Nonetheless, the anti-cataplectic activity of TAK-925 and ARN-776 is encouraging for the development of HCRTR2 agonists.

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP-importin α interactions.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

A Medicinal Chemistry-Driven Approach Identified the Sterol Isomerase EBP as the Molecular Target of TASIN Colorectal Cancer Toxins.

TASIN (Truncated APC-Selective Inhibitors) compounds are selectively toxic to colorectal cancer cells with APC mutations, although their mechanism of action remains unknown. Here, we found that TASINs inhibit three enzymes in the postsqualene cholesterol biosynthetic pathway including EBP, DHCR7, and DHCR24. Even though all three of these enzymes are required for cholesterol biosynthesis, only inhibition of the most upstream enzyme, EBP, led to cancer cell death via depletion of downstream sterols, an observation that was confirmed by genetic silencing of EBP. Pharmacologic inhibition or genetic silencing of either DHCR7 or DHCR24 had no impact on cell viability. By using photoaffinity probes to generate a relationship between chemical structure and probe competition, we identified compounds that selectively inhibit either EBP or DHCR7. These studies identify EBP, but not downstream enzymes in the cholesterol biosynthetic pathway, as a target in APC mutant colorectal cancer and also have implications for the clinical development of highly selective EBP inhibitors.

A convergent approach toward fidaxomicin: Syntheses of the fully glycosylated northern and southern fragments.

Efficient approaches that enable the synthesis of analogs of natural product antibiotics are needed to keep up with the emergence of multiply-resistant strains of pathogenic organisms. One promising candidate in this area is fidaxomicin, which boasts impressive in vitro anti-tubercular activity but has poor systemic bioavailability. We designed a flexible synthetic route to this target to enable the exploration of new chemical space and the future development of analogs with superior pharmacokinetics. We developed a robust approach to each of the key macrocyclic and sugar fragments, their union via stereoselective glycosylation, and a convergent late-stage macrolide formation with fully glycosylated fragments. Although we were able to demonstrate that the final Suzuki cross-coupling and ring-closing metathesis steps enabled macrocycle formation in the presence of the northern resorcylic rhamnoside and southern novioside sugars, these final steps were hampered by poor yields and the formation of the unwanted Z-macrocycle as the major stereoisomer.

Triazoles inhibit cholesterol export from lysosomes by binding to NPC1.

Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.

A biosynthetically inspired synthesis of (-)-berkelic acid and analogs.

We describe a complete account of our total synthesis and biological evaluation of (-)-berkelic acid and analogs. We delineate a synthetic strategy inspired by a potentially biomimetic union between the natural products spicifernin and pulvilloric acid. After defining optimal parameters, we executed a one-pot silver-mediated in situ dehydration of an isochroman lactol to methyl pulvillorate, the cycloisomerization of a spicifernin-like alkynol to the corresponding exocyclic enol ether, and a subsequent cycloaddition to deliver the tetracyclic core of berkelic acid. Our studies confirm that the original assigned berkelic acid structure is not stable and equilibrates into a mixture of 4 diastereomers, fully characterized by X-ray crystallography. In addition to berkelic acid, C22-epi-berkelic acid, and nor-berkelic acids, we synthesized C26-oxoberkelic acid analogs that were evaluated against human cancer cell lines. In contrast to data reported for natural berkelic acid, our synthetic material and analogs were found to be devoid of activity.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.

Synthesis and evaluation of analogs of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811, or AbeAdo) - An inhibitor of S-adenosylmethionine decarboxylase with antitrypanosomal activity.

We describe our efforts to improve the pharmacokinetic properties of a mechanism-based suicide inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), essential for the survival of the eukaryotic parasite Trypanosoma brucei responsible for Human African Trypanosomiasis (HAT). The lead compound, 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (1, also known as MDL 73811, or AbeAdo), has curative efficacy at a low dosage in a hemolymphatic model of HAT but displayed no demonstrable effect in a mouse model of the CNS stage of HAT due to poor blood-brain barrier permeation. Therefore, we prepared and evaluated an extensive set of analogs with modifications in the aminobutenyl side chain, the 5'-amine, the ribose, and the purine fragments. Although we gained valuable structure-activity insights from this comprehensive dataset, we did not gain traction on improving the prospects for CNS penetration while retaining the potent antiparasitic activity and metabolic stability of the lead compound 1.

Rifamycin Biosynthetic Congeners: Isolation and Total Synthesis of Rifsaliniketal and Total Synthesis of Salinisporamycin and Saliniketals A and B.

We describe the isolation, structure elucidation, and total synthesis of the novel marine natural product rifsaliniketal and the total synthesis of the structurally related variants salinisporamycin and saliniketals A and B. Rifsaliniketal was previously proposed, but not observed, as a diverted metabolite from a biosynthetic precursor to rifamycin S. Decarboxylation of rifamycin provides salinisporamycin, which upon truncation with loss of the naphthoquinone ring leads to saliniketals. Our synthetic strategy hinged upon a Pt(II)-catalyzed cycloisomerization of an alkynediol to set the dioxabicyclo[3.2.1]octane ring system and a fragmentation of an intermediate dihydropyranone to forge a stereochemically defined (E,Z)-dienamide unit. Multiple routes were explored to assemble fragments with high stereocontrol, an exercise that provided additional insights into acyclic stereocontrol during stereochemically complex fragment-assembly processes. The resulting 11-14 step synthesis of saliniketals then enabled us to explore strategies for the synthesis and coupling of highly substituted naphthoquinones or the corresponding naphthalene fragments. Whereas direct coupling with naphthoquinone fragments proved unsuccessful, both amidation and C-N bond formation tactics with the more electron-rich naphthalene congeners provided an efficient means to complete the first total synthesis of rifsaliniketal and salinisporamycin.

Synthesis and structure-activity studies of the V-ATPase inhibitor saliphenylhalamide (SaliPhe) and simplified analogs.

An efficient total synthesis of the potent V-ATPase inhibitor saliphenylhalamide (SaliPhe), a synthetic variant of the natural product salicylihalamide A (SaliA), has been accomplished aimed at facilitating the development of SaliPhe as an anticancer and antiviral agent. This new approach enabled facile access to derivatives for structure-activity relationship studies, leading to simplified analogs that maintain SaliPhe's biological properties. These studies will provide a solid foundation for the continued evaluation of SaliPhe and analogs as potential anticancer and antiviral agents.

Inhibition by cellular vacuolar ATPase impairs human papillomavirus uncoating and infection.

Several viruses, including human papillomaviruses, depend on endosomal acidification for successful infection. Hence, the multisubunit enzyme vacuolar ATPase (V-ATPase), which is mainly responsible for endosome acidification in the cell, represents an attractive target for antiviral strategies. In the present study, we show that V-ATPase is required for human papillomavirus (HPV) infection and that uncoating/disassembly but not endocytosis is affected by V-ATPase inhibition. The infection inhibitory potencies of saliphenylhalamide, a proven V-ATPase inhibitor, and its derivatives, as well as those of other V-ATPase inhibitors, were analyzed on different HPV types in relevant cell lines. Variation in the selectivity indices among V-ATPase inhibitors was high, while variation for the same inhibitor against different HPV subtypes was low, indicating that broad-spectrum anti-HPV activity can be provided.

Anticancer benzoxaboroles block pre-mRNA processing by directly inhibiting CPSF3.

A novel class of benzoxaboroles was reported to induce cancer cell death but the mechanism was unknown. Using a forward genetics platform, we discovered mutations in cleavage and polyadenylation specific factor 3 (CPSF3) that reduce benzoxaborole binding and confer resistance. CPSF3 is the endonuclease responsible for pre-mRNA 3'-end processing, which is also important for RNA polymerase II transcription termination. Benzoxaboroles inhibit this endonuclease activity of CPSF3 in vitro and also curb transcriptional termination in cells, which results in the downregulation of numerous constitutively expressed genes. Furthermore, we used X-ray crystallography to demonstrate that benzoxaboroles bind to the active site of CPSF3 in a manner distinct from the other known inhibitors of CPSF3. The benzoxaborole compound impeded the growth of cancer cell lines derived from different lineages. Our results suggest benzoxaboroles may represent a promising lead as CPSF3 inhibitors for clinical development.

Discovery and Optimization of N-Arylated Tetracyclic Dicarboximides That Target Primary Glioma Stem-like Cells.

We recently discovered a novel N-aryl tetracyclic dicarboximide MM0299 (1) with robust activity against glioma stem-like cells that potently and selectively inhibits lanosterol synthase leading to the accumulation of the toxic shunt metabolite 24(S),25-epoxycholesterol. Herein, we delineate a systematic and comprehensive SAR study that explores the structural space surrounding the N-aryl tetracyclic dicarboximide scaffold. A series of 100 analogs were synthesized and evaluated for activity against the murine glioma stem-like cell line Mut6 and for metabolic stability in mouse liver S9 fractions. This study led to several analogs with single-digit nanomolar activity in Mut6 glioblastoma cells that were metabolically stable in S9 fractions. In vivo pharmacokinetic analysis of selected analogs identified compound 52a (IC50 = 63 nM; S9 T1/2 > 240 min) which was orally available (39% plasma; 58% brain) and displayed excellent brain exposure. Chronic oral dosing of 52a during a 2-week tolerability study indicated no adverse effect on body weight nor signs of hematologic, liver, or kidney toxicity.

Thiophenyl Derivatives of Nicotinamide Are Metabolized by the NAD Salvage Pathway into Unnatural NAD Derivatives That Inhibit IMPDH and Are Toxic to Peripheral Nerve Cancers.

N-Pyridinylthiophene carboxamide (compound 21) displays activity against peripheral nerve sheath cancer cells and mouse xenografts by an unknown mechanism. Through medicinal chemistry, we identified a more active derivative, compound 9, and found that only analogues with structures similar to nicotinamide retained activity. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers.

Obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection.

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

Lessons and revelations from biomimetic syntheses.

Biomimetic synthesis describes the field of organic chemistry that aims to emulate the natural, biosynthetic processes toward natural products. As well as providing insight into how molecules are formed in nature, the benefits of this approach to total synthesis are numerous and extend beyond the gains typical of traditional synthesis. For example, using biosynthetic proposals to design a synthetic route can highlight alternative methods to the desired target. The pursuit of biomimetic syntheses also promotes the development of new reactions to prove or disprove a biosynthetic proposal or to unravel mechanistic implications of a proposed biosynthesis and can lead to the identification of new natural products. Here we look at some recent compelling examples and examine how biomimetic synthesis has led to the discovery of new procedures and principles that would not have been found by other approaches.

A room temperature copper catalyzed N-selective arylation of ß-amino alcohols with iodoanilines and aryl iodides.

An efficient method is described for the synthesis of N-(2-aminophenyl)-2-hydroxyethylamines via a copper catalyzed N-selective arylation of ß-amino alcohols with iodoanilines. The corresponding coupling products are useful intermediates for the synthesis of a variety of N-2-hydroxyethyl-substituted benzimidazoles, benzimidazolones, and iminobenzimidazoles. We found that 2-iodoaniline only arylates certain amino alcohols but not amines lacking a hydroxyl group. We also demonstrate the arylation of sterically demanding ß-amino alcohols, such as ephedrine and prolinol with aryl iodides at room temperature.

Structural insights for selective disruption of Beclin 1 binding to Bcl-2.

Stimulation of autophagy could provide powerful therapies for multiple diseases, including cancer and neurodegeneration. An attractive drug target for this purpose is Bcl-2, which inhibits autophagy by binding to the Beclin 1 BH3-domain. However, compounds that preclude Beclin 1/Bcl-2 binding might also induce apoptosis, which is inhibited by binding of Bcl-2 to BH3-domains of pro-apoptosis factors such as Bax. Here we describe the NMR structure of Bcl-2 bound to 35, a compound that we recently found to inhibit Beclin 1/Bcl-2 binding more potently than Bax/Bcl-2 binding. The structure shows that 35 binds at one end of the BH3-binding groove of Bcl-2. Interestingly, much of the 35-binding site is not involved in binding to Bcl-2 inhibitors described previously and mediates binding to Beclin 1 but not Bax. The structure suggests potential avenues to design compounds that disrupt Beclin 1/Bcl-2 binding and stimulate autophagy without inducing apoptosis.