Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential.

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.

Rebaudiosides T and U, minor C-19 xylopyranosyl and arabinopyranosyl steviol glycoside derivatives from Stevia rebaudiana (Bertoni) Bertoni.

Two diterpene glycosides were isolated from a commercial Stevia rebaudiana leaf extract. One was found to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(2-O-ß-d-xylopyranosyl-3-O-ß-d-glucopyranosyl- ß-d-glucopyranosyl) ester (rebaudioside T), whereas the other was determined to be 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-(6-O-α-l-arabinopyranosyl-ß-d-glucopyranosyl) ester (rebaudioside U). In addition, five C-19 sugar free derivatives were prepared and identified as follows: 13-[(2-O-α-l-rhamnopyranosyl-ß-d-glucopyranosyl)]oxy]kaur-16-en-19-oic acid (dulcoside A1); 13-[(2-O-ß-d-xylopyranosy-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]kaur-16-en-19-oic acid; 13-[(2-O-ß-d-xylopyranosyl-ß-d-glucopyranosyl-)oxy]kaur-16-en-19-oic acid; 13-[(2-O-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-xylopyranosyl-)oxy]kaur-16-en-19-oic acid (rebaudioside R1) and 13-[(2-O-6-deoxy-ß-d-glucopyranosyl-3-O-ß-d-glucopyranosyl-ß-d-glucopyranosyl)oxy]kaur-16-en-19-oic acid, respectively. Chemical structures were determined by NMR experiments. HPLC analyses were also useful to differentiate different steviol-C13 sugar substituent patterns by elution position.

SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A.

In order to expand the repertoire of antifungal compounds a novel, high-throughput phenotypic drug screen targeting fungal phosphatidylserine (PS) synthase (Cho1p) was developed based on antagonism of the toxin papuamide A (Pap-A). Pap-A is a cyclic depsipeptide that binds to PS in the membrane of wild-type Candida albicans, and permeabilizes its plasma membrane, ultimately causing cell death. Organisms with a homozygous deletion of the CHO1 gene (cho1ΔΔ) do not produce PS and are able to survive in the presence of Pap-A. Using this phenotype (i.e. resistance to Pap-A) as an indicator of Cho1p inhibition, we screened over 5,600 small molecules for Pap-A resistance and identified SB-224289 as a positive hit. SB-224289, previously reported as a selective human 5-HT1B receptor antagonist, also confers resistance to the similar toxin theopapuamide (TPap-A), but not to other cytotoxic depsipeptides tested. Structurally similar molecules and truncated variants of SB-224289 do not confer resistance to Pap-A, suggesting that the toxin-blocking ability of SB-224289 is very specific. Further biochemical characterization revealed that SB-224289 does not inhibit Cho1p, indicating that Pap-A resistance is conferred by another undetermined mechanism. Although the mode of resistance is unclear, interaction between SB-224289 and Pap-A or TPap-A suggests this screening assay could be adapted for discovering other compounds which could antagonize the effects of other environmentally- or medically-relevant depsipeptide toxins.

Diversity-oriented natural product platform identifies plant constituents targeting Plasmodium falciparum.

BACKGROUND: A diverse library of pre-fractionated plant extracts, generated by an automated high-throughput system, was tested using an in vitro anti-malarial screening platform to identify known or new natural products for lead development. The platform identifies hits on the basis of in vitro growth inhibition of Plasmodium falciparum and counter-screens for cytotoxicity to human foreskin fibroblast or embryonic kidney cell lines. The physical library was supplemented by early-stage collection of analytical data for each fraction to aid rapid identification of the active components within each screening hit. RESULTS: A total of 16,177 fractions from 1300 plants were screened, identifying several P. falciparum inhibitory fractions from 35 plants. Although individual fractions were screened for bioactivity to ensure adequate signal in the analytical characterizations, fractions containing less than 2.0 mg of dry weight were combined to produce combined fractions (COMBIs). Fractions of active COMBIs had EC50 values of 0.21-50.28 and 0.08-20.04 µg/mL against chloroquine-sensitive and -resistant strains, respectively. In Berberis thunbergii, eight known alkaloids were dereplicated quickly from its COMBIs, but berberine was the most-active constituent against P. falciparum. The triterpenoids α-betulinic acid and ß-betulinic acid of Eugenia rigida were also isolated as hits. Validation of the anti-malarial discovery platform was confirmed by these scaled isolations from B. thunbergii and E. rigida. CONCLUSIONS: These results demonstrate the value of curating and exploring a library of natural products for small molecule drug discovery. Attention given to the diversity of plant species represented in the library, focus on practical analytical data collection, and the use of counter-screens all facilitate the identification of anti-malarial compounds for lead development or new tools for chemical biology.

Solution structure of a sponge-derived cystine knot peptide and its notable stability.

A novel cystine knot peptide, asteropsin E (ASPE), was isolated from an Asteropus sp. marine sponge. The primary, secondary, and tertiary structures of ASPE were determined by high-resolution 2D NMR spectroscopy (900 MHz). With the exception of an N-terminal modification, ASPE shares properties with the previously reported asteropsins A-D, that is, the absence of basic residues, a highly acidic nature, conserved structurally important residues (including two cis-prolines), and a highly conserved tertiary structural framework. ASPE was found to be remarkably stable to gastrointestinal tract enzymes (chymotrypsin, elastase, pepsin, and trypsin) and to human plasma.

Asteropsins B-D, sponge-derived knottins with potential utility as a novel scaffold for oral peptide drugs.

BACKGROUND: Known linear knottins are unsuitable as scaffolds for oral peptide drug due to their gastrointestinal instability. Herein, a new subclass of knottin peptides from Porifera is structurally described and characterized regarding their potential for oral peptide drug development. METHODS: Asteropsins B-D (ASPB, ASPC, and ASPD) were isolated from the marine sponge Asteropus sp. The tertiary structures of ASPB and ASPC were determined by solution NMR spectroscopy and that of ASPD by homology modeling. RESULTS: The isolated asteropsins B-D, together with the previously reported asteropsin A (ASPA), compose a new subclass of knottins that share a highly conserved structural framework and remarkable stability against the enzymes in gastrointestinal tract (chymotrypsin, elastase, pepsin, and trypsin) and human plasma. CONCLUSION: Asteropsins can be considered as promising peptide scaffolds for oral bioavailability. GENERAL SIGNIFICANCE: The structural details of asteropsins provide essential information for the engineering of orally bioavailable peptides.

Configurational assignments of conformationally restricted bis-monoterpene hydroquinones: utility in exploration of endangered plants.

BACKGROUND: Endangered plant species are an important resource for new chemistry. Lindera melissifolia is native to the Southeastern U.S. and scarcely populates the edges of lakes and ponds. Quantum mechanics (QM) used in combination with NMR/ECD is a powerful tool for the assignment of absolute configuration in lieu of X-ray crystallography. METHODS: The EtOAc extract of L. melissifolia was subject to chromatographic analysis by VLC and HPLC. Spin-spin coupling constant (SSCC) were calculated using DFT at the MPW1PW91/6-31G(d,p) level for all staggered rotamers. ECD calculations employed Amber* force fields followed by PM6 semi-empirical optimizations. Hetero- and homo-nuclear coupling constants were extracted from 1D (1)H, E.COSY and HETLOC experiments. RESULTS: Two meroterpenoids, melissifolianes A (1) and B (2) were purified and their 2-D structures elucidated using NMR and HRESIMS. The relative configuration of 1 was established using the combination of NOE-based distance restraints and the comparisons of experimental and calculated SSCCs. The comparison of calculated and experimental ECD assigned the absolute configuration of 1. The relative configuration of a racemic mixture, melissifoliane B (2) was established utilizing J-based analysis combined with QM and NMR techniques.Conclusion Our study of the Lindera melissifolia metabolome exemplifies how new chemistry remains undiscovered among the numerous endangered plant species and demonstrates how analysis by ECD and NMR combined with various QM calculations is a sensible approach to support the stereochemical assignment of molecules with conformationally restricted conformations. GENERAL SIGNIFICANCE: QM-NMR/ECD combined approaches are of utility for unambiguous assignment of 3-D structures, especially with limited plant material and when a molecule is conformationally restricted. Conservation of an endangered plant species can be supported through identification of its new chemistry and utilization of that chemistry for commercial purposes.

Computationally assisted assignment of kahalalide Y configuration using an NMR-constrained conformational search.

Assignment of the absolute configuration of cyclic peptides frequently yields challenges, leaving one or more stereogenic centers unassigned due to small quantities of sample and the limited utility of Marfey's or other methods for assigning amino or hydroxy acids. Here, we report isolation of kahalalide Y (1) from Bryopsis pennata for the first time; in addition, the application of a combination of molecular modeling and NOE distance constraint calculations was utilized to determine the conformation of 1 and the absolute configuration of the final stereogenic center of 1. Using the Schrödinger suite, the structure of 1 was sketched in Maestro and minimized using the OPLS2005 force field in Macromodel. A conformational search was performed separately for structures having an R or S configuration at C-3 of the beta-hydroxy fatty acid subunit that completes the cyclic scaffold of 1, after which multiple minimizations for all generated conformers were carried out. The lowest energy conformers of R and S stereoisomers were then subjected to B3LYP geometry optimizations including solvent effects. The S stereoisomer was shown to be in excellent agreement with the NOE-derived distance constraints and hydrogen-bonding stability studies.

Asteropsin A: an unusual cystine-crosslinked peptide from porifera enhances neuronal Ca2+ influx.

BACKGROUND: Herein we report the discovery of a cystine-crosslinked peptide from Porifera along with high-quality spatial details accompanied by the description of its unique effect on neuronal calcium influx. METHODS: Asteropsin A (ASPA) was isolated from the marine sponge Asteropus sp., and its structure was independently determined using X-ray crystallography (0.87 angstroms) and solution NMR spectroscopy. RESULTS: An N-terminal pyroglutamate modification, uncommon cis proline conformations, and absence of basic residues helped distinguish ASPA from other cystine-crosslinked knot peptides. ASPA enhanced Ca2+ influx in murine cerebrocortical neuron cells following the addition of the Na+ channel activator veratridine but did not modify the oscillation frequency or amplitude of neuronal Ca2+ currents alone. Allosterism at neurotoxin site 2 was not observed, suggesting an alternative to the known Na+ channel interaction. CONCLUSIONS: Together with a distinct biological activity, the origin of ASPA suggests a new subclass of cystine-rich knot peptides associated with Porifera. GENERAL SIGNIFICANCE: The discovery of ASPA represents a distinctive addition to an emerging subclass of cystine-crosslinked knot peptides from Porifera.

Natural product studies of U.S. endangered plants: volatile components of Lindera melissifolia (Lauraceae) repel mosquitoes and ticks.

The number of endangered plant species in the U.S. is significant, yet studies aimed towards utilizing these plants are limited. Ticks and mosquitoes are vectors of significant pathogenic diseases of humans. Repellents are critical means of personal protection against biting arthropods and disease transmission. The essential oil and solvent extracts from Lindera melissifolia (Walt.) Blume (Lauraceae) (pondberry) drupes were gathered and analyzed by GC and GC-MS. The essential oil obtained from this endangered plant showed a significant dose dependent repellency of ticks and a moderate mosquito repellent effect while the subsequent hexanes extract was completely ineffective. Fractional freezing enriched the tick repellent components of the essential oil. Several known tick repellent components were recognized by the GC-MS comparison of the resulting fractions and ß-caryophyllene, α-humulene, germacrene D and ß-elemene warrant evaluations for tick repellency. Identifying pondberry as a potential renewable source for a broad spectrum repellent supports efforts to conserve similar U.S. endangered or threatened plant species.

Reducing oyster-associated bacteria levels using supercritical fluid CO2 as an agent of warm pasteurization.

An innovative approach to Post-Harvest Processing (PHP) of oysters is introduced focusing on the effects of supercritical carbon dioxide (scCO(2)) on bacterial contaminants trapped in the digestive system of oysters. Oysters were exposed to scCO(2) under two conditions: (1) 100 bar and 37 degrees C for 30 min and (2) 172 bar and 60 degrees C for 60 min. Using FDA standard guidelines for food analysis, variations in the Aerobic Plate Count (APC) were assessed. It was established that exposing oysters to CO(2) at 100 bar and 37 degrees C for 30 min and at 172 bar and 60 degrees C for 60 min induced 2-log and 3-log reductions in the APC respectively. The decrease in the microbial load as a result of treatment with scCO(2) was found to be significant (P=0.002). A release of adductor muscles from the shell was noted in oysters treated at 172 bar and 60 degrees C for 60 min; this was not the case for oysters treated at 100 bar and 37 degrees C for 30 min. A blind study allowing sensory analysis of treated vs. untreated oysters was also completed and no significant change in the physical appearance, smell, or texture was recorded. In this paper, we also report the effect of scCO(2) on several bacterial isolates, including a referenced ATCC strain of a non-pathogenic Vibrio (Vibrio fischeri) as well as several other bacterial isolates cultured from oyster' tissues and found to share biochemical features common to pathogenic Vibrio strains. A complete inactivation (minimum 7-log reduction) was achieved with these latter bacterial isolates. A 6-log reduction was observed with V. fischeri.

Antiviral and anticancer optimization studies of the DNA-binding marine natural product aaptamine.

Aaptamine has potent cytotoxicity that may be explained by its ability to intercalate DNA. Aaptamine was evaluated for its ability to bind to DNA to validate DNA binding as the primary mechanism of cytotoxicity. Based on UV-vis absorbance titration data, the K(obs) for aaptamine was 4.0 (+/-0.2) x 10(3) which was essentially equivalent to the known DNA intercalator N-[2-(diethylamino)ethyl]-9-aminoacridine-4-carboxamide. Semi-synthetic core modifications were performed to improve the general structural diversity of known aaptamine analogs and vary its absorption characteristics. Overall, 26 aaptamine derivatives were synthesized which consisted of a simple homologous range of mono and di-N-alkylations as well as some 9-O-sulfonylation and bis-O-isoaaptamine dimer products. Each product was evaluated for activity in a variety of whole cell and viral assays including a unique solid tumor disk diffusion assay. Details of aaptamine's DNA-binding activity and its derivatives' whole cell and viral assay results are discussed.

Latrunculin with a highly oxidized thiazolidinone ring: structure assignment and actin docking.

A new latrunculin, oxalatrunculin B (3), was isolated from Red Sea sponge Negombata corticata. Extensive spectroscopic analysis revealed an unprecedented heterocycle in which the rare thiazolidinone ring found in latrunculins was oxidized with three additional oxygens. An actin polymerization inhibition assay agreed with MM-PBSA free energy calculations that 3 binds more weakly than latrunculin B to actin. Significant antifungal and anticancer activity of 3 was found, suggesting an alternate target in addition to actin for latrunculin bioactivity.

Nature's bounty - drug discovery from the sea.

With ∼ 40 years of research completed after the development of self-contained underwater breathing apparatus, drug discovery opportunities in the sea are still too numerous to count. Since the FDA approval of the direct-from-the-sea calcium channel blocker ziconotide, marine natural products have been validated as a source for new medicines. However, the demand for natural products is extremely high due to the development of high-throughput assays and this bottleneck has created the need for an intense focus on increasing the rate of isolating and elucidating the structures of new bioactive secondary metabolites. In addition to highlighting the drug discovery potential of the marine environment, this review discusses several of the pressing needs to increase the rate of drug discovery in marine natural products, and describes some of the work and new technologies that are contributing in this regard.

Modification at the C9 position of the marine natural product isoaaptamine and the impact on HIV-1, mycobacterial, and tumor cell activity.

As part of an investigation to generate optimized drug leads from marine natural pharmacophores for the treatment of neoplastic and infectious diseases, a series of novel isoaaptamine analogs were prepared by coupling acyl halides to the C9 position of isoaaptamine (2) isolated from the Aaptos sponge. This library of new semisynthetic products was evaluated for biological activity against HIV-1, Mtb, AIDS-OI, tropical parasitic diseases, and cancer. Compound 4 showed potent activity against HIV-1 (EC(50) 0.47microg/mL), compound 19 proved to possess remarkable activity against Mycobacterium intracellulare with an IC(50) and MIC value of 0.15 and 0.31microg/mL, while compounds 4 and 17 possessed anti-leishmanial activity with IC(50) values of 0.1 and 0.4microg/mL, respectively. Compounds 16 and 17 showed antimalarial activity with EC(50) values of 230 and 240ng/mL, respectively, and compound 14 exhibited an EC(50) of 0.05microM against the Leukemia cell line K-562.

Zebra mussel antifouling activity of the marine natural product aaptamine and analogs.

Several aaptamine derivatives were selected as potential zebra mussel (Dreissena polymorpha) antifoulants because of the noteworthy absence of fouling observed on Aaptos sponges. Sponges of the genus Aaptos collected in Manado, Indonesia consistently produce aaptamine-type alkaloids. To date, aaptamine and its derivatives have not been carefully evaluated for their antifoulant properties. Structure-activity relationship studies were conducted using several aaptamine derivatives in a zebra mussel antifouling assay. From these data, three analogs have shown significant antifouling activity against zebra mussel attachment. Aaptamine, isoaaptamine, and the demethylated aaptamine compounds used in the zebra mussel assay produced EC(50) values of 24.2, 11.6, and 18.6 microM, respectively. In addition, neither aaptamine nor isoaaptamine produced a phytotoxic response (as high as 300 microM) toward a nontarget organism, Lemna pausicostata, in a 7-day exposure. The use of these aaptamine derivatives from Aaptos sp. as potential environmentally benign antifouling alternatives to metal-based paints and preservatives is significant, not only as a possible control of fouling organisms, but also to highlight the ecological importance of these and similar biochemical defenses.

Structural activity relationship studies of zebra mussel antifouling and antimicrobial agents from verongid sponges.

Several dibromotyramine derivatives including moloka'iamine were selected as potential zebra mussel (Dreissena polymorpha) antifoulants due to the noteworthy absence of fouling observed on sponges of the order Verongida. Sponges of the order Verongida consistently produce these types of bromotyrosine-derived secondary metabolites. Previously reported antifouling data for the barnacle Balanus amphitrite(EC50 = 12.2 microM) support the results reported here that the compound moloka'iamine may be a potential zebra mussel antifoulant compound (EC50 = 10.4 microM). The absence of phytotoxic activity of the compound moloka'iamine toward Lemna pausicostata and, most importantly, the compound's significant selectivity against macrofouling organisms such as zebra mussels suggest the potential utility of this compound as a naturally derived antifoulant lead.