Cynthia Jeffries

Automated high-throughput system to fractionate plant natural products for drug discovery.

The development of an automated, high-throughput fractionation procedure to prepare and analyze natural product libraries for drug discovery screening is described. Natural products obtained from plant materials worldwide were extracted and first prefractionated on polyamide solid-phase extraction cartridges to remove polyphenols, followed by high-throughput automated fractionation, drying, weighing, and reformatting for screening and storage. The analysis of fractions with UPLC coupled with MS, PDA, and ELSD detectors provides information that facilitates characterization of compounds in active fractions. Screening of a portion of fractions yielded multiple assay-specific hits in several high-throughput cellular screening assays. This procedure modernizes the traditional natural product fractionation paradigm by seamlessly integrating automation, informatics, and multimodal analytical interrogation capabilities.

UPLC-MS-ELSD-PDA as a powerful dereplication tool to facilitate compound identification from small-molecule natural product libraries.

The generation of natural product libraries containing column fractions, each with only a few small molecules, using a high-throughput, automated fractionation system, has made it possible to implement an improved dereplication strategy for selection and prioritization of leads in a natural product discovery program. Analysis of databased UPLC-MS-ELSD-PDA information of three leads from a biological screen employing the ependymoma cell line EphB2-EPD generated details on the possible structures of active compounds present. The procedure allows the rapid identification of known compounds and guides the isolation of unknown compounds of interest. Three previously known flavanone-type compounds, homoeriodictyol (1), hesperetin (2), and sterubin (3), were identified in a selected fraction derived from the leaves of Eriodictyon angustifolium. The lignan compound deoxypodophyllotoxin (8) was confirmed to be an active constituent in two lead fractions derived from the bark and leaves of Thuja occidentalis. In addition, two new but inactive labdane-type diterpenoids with an uncommon triol side chain were also identified as coexisting with deoxypodophyllotoxin in a lead fraction from the bark of T. occidentalis. Both diterpenoids were isolated in acetylated form, and their structures were determined as 14S,15-diacetoxy-13R-hydroxylabd-8(17)-en-19-oic acid (9) and 14R,15-diacetoxy-13S-hydroxylabd-8(17)-en-19-oic acid (10), respectively, by spectroscopic data interpretation and X-ray crystallography. This work demonstrates that a UPLC-MS-ELSD-PDA database produced during fractionation may be used as a powerful dereplication tool to facilitate compound identification from chromatographically tractable small-molecule natural product libraries.

Heterobiaryl Human Immunodeficiency Virus Entry Inhibitors

Previously disclosed HIV (human immunodeficiency virus) attachment inhibitors, exemplified by BMS 806 (formally BMS378806, 1), are characterized by a substituted indole or azaindole ring linked to a benzoylpiperazine via a ketoamide or sulfonamide group. In the present report, we describe the discovery of a novel series of potent HIV entry inhibitors in which the indole or azaindole ring of previous inhibitors is replaced by a heterobiaryl group. Several of these analogues exhibited IC(50) values of less than 5 nM in a pseudotyped antiviral assay, and compound 13k was demonstrated to exhibit potency and selectivity similar to those of 1 against a panel of clinical viral isolates. Moreover, current structure-activity relationship studies of these novel biaryl gp120 inhibitors revealed that around the biaryl, a fine crevice might exist in the gp120 binding site. Taken in sum, these data reveal a hitherto unsuspected flexibility in the structure-activity relationships for these inhibitors and suggest new avenues for exploration and gp120 inhibitor design.

Synthesis and evaluation of sulfonylnitrophenylthiazoles (SNPTs) as thyroid hormone receptor-coactivator interaction inhibitors.

We previously identified a series of methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its coactivators. MSNBs inhibit coactivator binding through irreversible modification of cysteine 298 of the thyroid hormone receptor (TR). Although MSNBs have better pharmacological features than our first generation inhibitors (β-aminoketones), they contain a potentially unstable ester linkage. Here we report the bioisosteric replacement of the ester linkage with a thiazole moiety, yielding sulfonylnitrophenylthiazoles (SNPTs). An array of SNPTs representing optimal side chains from the MSNB series was constructed using parallel chemistry and evaluated to test their antagonism of the TR-coactivator interaction. Selected active compounds were evaluated in secondary confirmatory assays including regulation of thyroid response element driven transcription in reporter constructs and native genes. In addition the selected SNPTs were shown to be selective for TR relative to other nuclear hormone receptors (NRs).

Development of BODIPY FL Vindoline as a Novel and High-Affinity Pregnane X Receptor Fluorescent Probe

The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows it to bind many drugs and drug leads, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether chemicals or drugs bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. On the basis of our previously characterized 4 (BODIPY FL vinblastine), a high-affinity PXR probe, we developed 20 (BODIPY FL vindoline) and showed that it is a novel and potent PXR fluorescent probe with Kd of 256 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay with PXR. By using 20 (BODIPY FL vindoline) in the PXR TR-FRET assay, we obtained a more than 7-fold signal-to-background ratio and high signal stability (signal was stable for at least 120 min, and Z′-factor > 0.85 from 30 to 240 min). The assay can tolerate DMSO up to 2%. This assay has been used to evaluate a panel of PXR ligands for their PXR-binding affinities. The performance of 20 (BODIPY FL vindoline) in the PXR TR-FRET assay makes it an ideal PXR fluorescent probe, and the newly developed PXR TR-FRET assay with 20 (BODIPY FL vindoline) as a fluorescent probe is suitable for high-throughput screening to identify PXR-binding ligands.

Selective Inhibitors of Human Liver Carboxylesterase Based on a β-Lapachone Scaffold: Novel Reagents for Reaction Profiling

Carboxylesterases (CEs) are ubiquitous enzymes that are responsible for the metabolism of xenobiotics, including drugs such as irinotecan and oseltamivir. Inhibition of CEs significantly modulates the efficacy of such agents. We report here that β-lapachone is a potent, reversible CE inhibitor with Ki values in the nanomolar range. A series of amino and phenoxy analogues have been synthesized, and although the former are very poor inhibitors, the latter compounds are highly effective in modulating CE activity. Our data demonstrate that tautomerism of the amino derivatives to the imino forms likely accounts for their loss in biological activity. A series of N-methylated amino derivatives, which are unable to undergo such tautomerism, were equal in potency to the phenoxy analogues and demonstrated selectivity for the liver enzyme hCE1. These specific inhibitors, which are active in cell culture models, will be exceptionally useful reagents for reaction profiling of esterified drugs in complex biological samples.

Novel vitexin-inspired scaffold against leukemia

Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in children. Up to a quarter of ALL patients relapse and face poor prognosis. To identify new compound leads, we conducted a phenotypic screen using terrestrial natural product (NP) fractions against immortalized ALL cellular models. We identified vitexin, a flavonoid, as a promising hit with biological activity (EC50 = 30 μM) in pre-B cell ALL models with no toxicity against normal human tissue (BJ cells) at the tested concentrations. To develop more potent compounds against ALL and elucidate its potential mode of action, a vitexin-inspired compound library was synthesized. Thus, we developed an improved and scalable protocol for the direct synthesis of 4-quinolone core heterocycles containing an N-sulfonamide using a one-pot condensation reaction protocol. The newly generated compounds represent a novel molecular scaffold against ALL as exemplified by compounds 13 and 15, which demonstrated EC50 values in the low micromolar range (0.3-10 μM) with little to no toxicity in normal cellular models. Computational studies support the hypothesis that these compounds are potential CDK inhibitors. The compounds induced apoptosis, caused cell arrest at G0/G1 and G2/M, and induced ROS in cancer cells.

Tissue Penetration of a Novel Spectinamide Antibiotic for the Treatment of Tuberculosis.

The in vivo biodistribution and pharmacokinetics of 1329, a novel spectinamide antibiotic with anti-tubercular activity, were studied during intravenous administration of an tritium-labeled compound for nine consecutive, 12-hourly doses to rats. Serial blood samples were collected after the first and the eighth dose, and major organs and tissues were collected 1 h after the ninth dose. Urinary and fecal excretion was monitored throughout the dosing period. Radioactivity in the collected samples was assessed by scintillation counting. During the course of treatment, 86.6% of the administered radioactivity was recovered in urine, feces, organs, and muscle tissue. Urinary excretion was the major route of elimination, with 70% of radioactivity recovered from urine and 12.6% from feces. The time profiles of radioactivity in serum after the first and the eighth dose were identical for the first 2 h post-dose, with similar Cmax (3.39 vs. 3.55 mCi/L) and AUC0−τ (5.08 vs. 5.17 mCi • h/L), indicating no substantial accumulation of 1329 during multiple dosing. Radioactivity in major target organs for pulmonary tuberculosis infection, the lungs and spleen, was 2.79- and 3.06-fold higher than in the blood. Similarly, the intracellular uptake of 1329 into macrophages was sixfold higher than for streptomycin. Overall, these observations suggest biodistribution properties favorable for targeting pulmonary tuberculosis infections.

Radiosynthesis of antitumor spliceosome modulators

A set of novel antitumor agents (the sudemycins) has recently been described that are analogs of the natural product FR901464. We report the radiosynthesis of two of these antitumor drug lead compounds, using a three step procedure: (1) ester hydrolysis, (2) Lindlars catalyst/tritium gas to give a (S,Z)-4-acetoxypent-2-enoic acid derivative, and finally (3) amide bond formation. These labeled analogs are useful in developing a better understanding of the pharmacological properties of this new class of therapeutic lead compounds.

Discovery of an Orally Bioavailable Inhibitor of Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously reported the discovery, validation, and structure-activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1-UBE2M interaction. We demonstrated that compound 7 inhibits both the DCN1-UBE2M protein-protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound 7 exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CLint = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (67). Compound 67 retains the favorable biochemical and cellular activity of compound 7 but is significantly more stable both in vitro and in vivo. Compound 67 is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1-UBE2M interaction on the NEDD8/CUL pathway.