Integrated biological—physicochemical system for the identification of antitumor compounds in fermentation broths

Hook, Derek J.; More, Carolee F.; Yacobucci, Joseph J.; Dubay, George R.; O'Connor, S. E.

doi:10.1016/s0021-9673(01)94625-4

Cited by 31 publications

(12 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Inclusion of an NS5A-resistant cell line in the experiments to demonstrate cross-resistance was key to confirming relatedness to BMS-824 and allowing us to identify the peak fractions of interest. A powerful aspect of the HPLC biogram methodology lies in the ability to use a functional assay to detect biologically active substances in crude matrices that are not readily detectable by physical methods such as, for example, HPLC-UV (5,8). We began the present study by incubating BMS-824 in HCV replicon assay medium prior to isolation experiments.…”

Section: Discussionmentioning

confidence: 99%

“…A 100-l aliquot of the supernatant was subjected to HPLC fractionation. The HPLC (C 18 ) conditions used included an Agilent HP-1100, a Waters X-Terra 5-m (C 18 ) column (4.6 by 150 mm), a mobile phase consisting of a 0.01% trifluoroacetic acid-acetonitrile gradient (8), a flow rate of 1.2 ml/min, and 254-nm UV detection. For the HPLC biogram (replicon assay) analyses, fractions were collected in Beckman 96-deep-well plates using a Gilson 215 liquid handler and dried under vacuum using a Savant SpeedVac.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Discovery of Potent Hepatitis C Virus NS5A Inhibitors with Dimeric Structures

Lemm

Leet

O’Boyle

et al. 2011

Antimicrob Agents Chemother

View full text Add to dashboard Cite

The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translated into an in vivo effect in proof-of-concept clinical trials. Although the 50% effective concentration (EC 50 ) of the initial lead, the thiazolidinone BMS-824, was ϳ10 nM in the replicon assay, it underwent transformation to other inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, including the EC 50 , the drug concentration required to reduce cell growth by 50% (CC 50 ), and the resistance profile, however, remained unchanged, triggering an investigation to identify the biologically active species. Highperformance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated in medium revealed that the most active fractions could readily be separated from the parental compound and retained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes in compound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to be discussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors provided the foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in the identification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.Hepatitis C virus (HCV) is the major causative agent of non-A, non-B hepatitis worldwide, which affects more than 3% of the world's population. Of those infected with HCV, ϳ70% proceed to a chronic state which can lead to severe liver diseases, including fibrosis, cirrhosis, or hepatocellular carcinoma (1, 7). There is currently no vaccine against HCV and no generally effective therapy for all HCV genotypes. The current optimal therapy is pegylated alpha interferon in combination with ribavirin, a regimen associated with significant side effects and limited efficacy in the most prevalent patient population, consisting of genotype 1 (4). Therefore, there is an urgent need for the development of more effective, HCV-specific antiviral therapies with fewer side effects.In the search for more efficacious, safer HCV therapies, the most actively pursued antiviral targets have been the NS3 protease and NS5B RNA-dependent RNA polymerase, both essential enzymes for the replication of HCV (2,11,12). Exciting progress has been demonstrated in clinical trials with multiple HCV NS3 serine protease inhibitors, as well as with both nucleoside and nonnucleoside po...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Discovery of Potent Hepatitis C Virus NS5A Inhibitors with Dimeric Structures

Lemm

Leet

O’Boyle

et al. 2011

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…The HPLC biogram methodology has its origins in bioactive natural products discovery programs, such as in screening for antitumor agents and antibiotics in microbial fermentation broths. 1 Similar methodology has also been used for the analysis of combinatorial library mixtures. 2 In the present context, we further extend the use of biogram methodology to chemical validation of small-molecule screening hits and, in many cases, further investigation of bioactive components in key samples having unexplained or poorly reproducible biological activity.…”

Section: Resultsmentioning

confidence: 99%

HPLC Biogram Analysis: A Powerful Tool Used for Hit Confirmation in Early Drug Discovery

et al. 2015

View full text Add to dashboard Cite

High-performance liquid chromatography (HPLC) biogram methodology is a powerful pharmaceutical screening hit confirmation strategy that couples analytical HPLC data with functional bioassay data. It is used primarily for screening hit chemical validation and triaging in support of early phase discovery programs and enables further investigation of the source of bioactivity in screening hits. The process combines semi-preparative separation technologies, automated compound handling and distribution, high-throughput biological screening, and informatics tools. The final output is an HPLC retention time versus bioactivity graphical overlay report. In this manner, biograms allow the analyst to determine which component in the sample is responsible for the biological activity, enabling decision making toward chemotype selection and prioritization from a pool of potential candidates. Another powerful aspect of the biogram assay lies in its utility in investigating biological activity in atypical samples, such as degraded samples or mixtures, for detection of minor active impurities or in addressing lot-to-lot activity discrepancies for a given test compound. Biograms are employed to track, isolate, and identify the source of biological activity in such samples, often yielding important information for program decisions.

show abstract

“…To identify an active component, a mixture sample needs to be fractionated, which consists of connecting a microscale fraction collector to an LC-UV-MS system and subjecting the collected fractions to various assays to reveal which peak is the active component ( Figure 3). Hook et al, 11,12 researchers at Bristol Myers Company, described their micro-fractionation process. They connected the fraction collector to HPLC-UV or HPLC-UV-MS and used microtiter plates to collect fractions that were then subjected to HTS using robotic systems.…”

Section: Micro-fractionation For Active Peak Identificationmentioning

confidence: 99%

“…A systematic dereplication method for crude extract samples that can be combined with HTS has been developed using LC-UV-(MS). 11,12 Dereplication was carried out in combination with microbiological study, such as identifying producer microbes by morphology and gene analysis, and sometimes used comprehensively in combination with various informatics techniques. 13 Currently, the term dereplication is used in a broader sense in the screening of natural products mixtures, as shown in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

Dereplication of microbial extracts and related analytical technologies

Itô¹,

Masubuchi²

2014

J Antibiot

View full text Add to dashboard Cite

Natural products still continue to have an important role as a resource of various biologically active substances. Dereplication is a key process in natural product screening that analyzes the extracts of microbial fermentation broths or plant samples. In this review article, we describe and discuss the analytical techniques of dereplication and related technologies in the following sections: 1. Direct detection from microbial colonies. 2. Ultra high performance liquid chromatography (UHPLC)-MS profiling for library construction. 3. Micro-fractionation to identify active peaks. 4. Quantification of small-amount compounds. 5. Structure identification from small amounts. Using these techniques, the desired compound in the mixture library can be rapidly identified.

show abstract

Integrated biological—physicochemical system for the identification of antitumor compounds in fermentation broths

Cited by 31 publications

References 9 publications

Discovery of Potent Hepatitis C Virus NS5A Inhibitors with Dimeric Structures

Discovery of Potent Hepatitis C Virus NS5A Inhibitors with Dimeric Structures

HPLC Biogram Analysis: A Powerful Tool Used for Hit Confirmation in Early Drug Discovery

Dereplication of microbial extracts and related analytical technologies

Contact Info

Product

Resources

About