Publications by authors named "Pan F Chan"

13 Publications

  • Page 1 of 1

Bimodal Actions of a Naphthyridone/Aminopiperidine-Based Antibacterial That Targets Gyrase and Topoisomerase IV.

Biochemistry 2019 11 28;58(44):4447-4455. Epub 2019 Oct 28.

VA Tennessee Valley Healthcare System , Nashville , Tennessee 37212 , United States.

Gyrase and topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore, it is critical to identify new agents that maintain activity against fluoroquinolone-resistant strains. One approach is to develop non-fluoroquinolone drugs that also target gyrase and topoisomerase IV but interact differently with the enzymes. This has led to the development of the "novel bacterial topoisomerase inhibitor" (NBTI) class of antibacterials. Despite the clinical potential of NBTIs, there is a relative paucity of data describing their mechanism of action against bacterial type II topoisomerases. Consequently, we characterized the activity of GSK126, a naphthyridone/aminopiperidine-based NBTI, against a variety of Gram-positive and Gram-negative bacterial type II topoisomerases, including gyrase from and gyrase and topoisomerase IV from and . GSK126 enhanced single-stranded DNA cleavage and suppressed double-stranded cleavage mediated by these enzymes. It was also a potent inhibitor of gyrase-catalyzed DNA supercoiling and topoisomerase IV-catalyzed decatenation. Thus, GSK126 displays a similar bimodal mechanism of action across a variety of species. In contrast, GSK126 displayed a variable ability to overcome fluoroquinolone resistance mutations across these same species. Our results suggest that NBTIs elicit their antibacterial effects by two different mechanisms: inhibition of gyrase/topoisomerase IV catalytic activity or enhancement of enzyme-mediated DNA cleavage. Furthermore, the relative importance of these two mechanisms appears to differ from species to species. Therefore, we propose that the mechanistic basis for the antibacterial properties of NBTIs is bimodal in nature.
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http://dx.doi.org/10.1021/acs.biochem.9b00805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450530PMC
November 2019

Structure-guided design of antibacterials that allosterically inhibit DNA gyrase.

Bioorg Med Chem Lett 2019 06 22;29(11):1407-1412. Epub 2019 Mar 22.

GlaxoSmithKline, Collegeville, PA 19426, USA. Electronic address:

A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.
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http://dx.doi.org/10.1016/j.bmcl.2019.03.029DOI Listing
June 2019

Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase.

ACS Infect Dis 2019 04 28;5(4):570-581. Epub 2019 Feb 28.

VA Tennessee Valley Healthcare System , 1310 24th Avenue S. , Nashville , Tennessee 37212 , United States.

Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC ≈ 0.047 μM) and relaxation of positively supercoiled substrates (IC ≈ 0.6 μM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound.
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http://dx.doi.org/10.1021/acsinfecdis.8b00315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461504PMC
April 2019

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening.

Nat Commun 2017 07 17;8:16081. Epub 2017 Jul 17.

GlaxoSmithKline, 830 Winter Street, Waltham, Massachusetts 02451, USA.

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.
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http://dx.doi.org/10.1038/ncomms16081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520047PMC
July 2017

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

Proc Natl Acad Sci U S A 2017 05 15;114(22):E4492-E4500. Epub 2017 May 15.

Antibacterial Discovery Performance Unit, Infectious Diseases Therapy Area Unit, GlaxoSmithKline, Collegeville, PA 19426;

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.
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http://dx.doi.org/10.1073/pnas.1700721114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465892PMC
May 2017

Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin.

Nat Commun 2015 Dec 7;6:10048. Epub 2015 Dec 7.

Antibacterial Discovery Performance Unit, Infectious Diseases, Therapy Area Unit, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426-0989, USA.

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.
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http://dx.doi.org/10.1038/ncomms10048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686662PMC
December 2015

Crystallization and initial crystallographic analysis of covalent DNA-cleavage complexes of Staphyloccocus aureus DNA gyrase with QPT-1, moxifloxacin and etoposide.

Acta Crystallogr F Struct Biol Commun 2015 Oct 23;71(Pt 10):1242-6. Epub 2015 Sep 23.

Platform Technology and Science, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, England.

Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.
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http://dx.doi.org/10.1107/S2053230X15015290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601586PMC
October 2015

Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance.

Nat Struct Mol Biol 2010 Sep 29;17(9):1152-3. Epub 2010 Aug 29.

Platform Technology and Science, GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire, UK.

Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.
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http://dx.doi.org/10.1038/nsmb.1892DOI Listing
September 2010

High-yield production and characterization of biologically active GST-tagged human topoisomerase IIα protein in insect cells for the development of a high-throughput assay.

Protein Expr Purif 2011 Apr 13;76(2):165-72. Epub 2010 Aug 13.

Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Harlow, Essex, UK.

DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIα proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIα using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIα was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIα inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ≥ 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.
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http://dx.doi.org/10.1016/j.pep.2010.08.001DOI Listing
April 2011

Type IIA topoisomerase inhibition by a new class of antibacterial agents.

Nature 2010 Aug 4;466(7309):935-40. Epub 2010 Aug 4.

Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
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http://dx.doi.org/10.1038/nature09197DOI Listing
August 2010

Characterization of a novel fucose-regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae.

J Bacteriol 2003 Mar;185(6):2051-8

Microbial, Musculoskeletal and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.

The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150135PMC
http://dx.doi.org/10.1128/jb.185.6.2051-2058.2003DOI Listing
March 2003

Novel antibacterials: a genomics approach to drug discovery.

Curr Drug Targets Infect Disord 2002 Dec;2(4):291-308

Department of Microbiology. Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426-0989, USA.

The appearance of antibiotic resistant pathogens, including vancomycin resistant Staphylococcus aureus, in the clinic has necessitated the development of new antibiotics. The golden age of antibiotic discovery, in which potent selective compounds were readily extracted from natural product extracts is over and novel approaches need to be implemented to cover the therapeutic shortfall. The generation of huge quantities of bacterial sequence data has allowed the identification of all the possible targets for therapeutic intervention and allowed the development of screens to identify inhibitors. Here, we described a number of target classes in which genomics has contributed to its identification. As a result of analyzing sequence data, all of the tRNA synthetases and all of the two-component signal transduction systems were readily isolated; which would not have been easily identified if whole genome sequences were not available. Fatty acid biosynthesis is a known antibacterial target, but genomics showed which genes in that pathway had the appropriate spectrum to be considered as therapeutic targets. Genes of unknown function may seem untractable targets, but if those that are broad spectrum and essential are identified, it becomes valuable to invest time and effort to determine their cellular role. In addition, we discuss the role of genomics in developing technologies that assist in the discovery of new antibiotics including microarray gridding technology. Genomics can also increase the chemical diversity against which the novel targets can be screened.
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http://dx.doi.org/10.2174/1568005023342227DOI Listing
December 2002