Molecular basis of DNA gating by topo IV and gyrase and its inhibition by antimicrobial drugs

Lead Research Organisation: St George's, University of London
Department Name: Basic Medical Sciences

Abstract

Most life-forms whether humans, mammals, yeast or bacteria have a DNA genome. The DNA consists of two strands that wrap around each other to form the iconic DNA helix. During DNA replication, the helix must be unwound to allow copying and chromosome segregation. This process generates DNA supercoils and DNA tangles that can block replication. Topoisomerases solve the 'unwinding problem' by introducing transient single- or double-stranded breaks into DNA that dissipate supercoils and allow DNA synthesis to proceed. It is not surprising that topoisomerases are essential enzymes but they also turn out to be vital targets of antibacterial and anticancer drugs that inhibit cell growth. We aim to use biochemical and X-ray crystallographic approaches to study two bacterial topoisomerases called topo IV and gyrase. We wish to understand how these proteins act at the molecular level and how clinically important drugs such as fluoroquinolones target their functions. The work will focus on the well-characterised topo IV and gyrase from Streptococcus pneumoniae, a dangerous human pathogen that causes life-threatening pneumonia and meningitis. However the research has wider applications to other bacterial and microbial systems. Progress in this key area should aid development of new drugs that work against antibiotic-resistant organisms, a growing threat to our healthcare system.

Technical Summary

Topo IV and gyrase mediate chromosome unlinking and DNA supercoiling and are targeted by clinically important antibacterial quinolones and other agents. Each enzyme transports a DNA duplex (the T segment) through a DNA gate made in a second DNA helix (the G-gate) in a process that involves the reversible formation of a covalent enzyme-DNA intermediate called the cleavage complex. In recent advances, we have determined the X-ray crystal structures of cleavage complexes of Streptococcus pneumoniae topo IV formed with quinolone and quinazolinediones. Building on these and other structures and new biochemical tools, we aim to examine how DNA is gated through the DNA enzyme complex and how novel drugs interfere with this process. The work will involve a mutidisciplinary approach employing biochemical, X-ray structure, EM and SAXS approaches. Completion of the study will provide new insights on DNA gating during catalysis by topo IV and gyrase and its arrest by anti-topoisomerase therapeutics.

Planned Impact

The pursuit of excellent basic research and its translation to drugs is an important feature of the proposal. The major beneficiaries who will be interested in or will benefit from the research are:

The pharmaceutical industry developing new antibacterial topo IV/gyrase inhibitors as anti-infectives

Microbiologists and drug developers interested in combatting the mechanisms of drug resistance

Structural biologists and nano-engineers studying molecular machines

A range of scientific disciplines with a focus on DNA-protein interactions

The very young and elderly with pneumococcal disease

Society at large- through effective disease management reducing the societal and economic costs of healthcare and hospitalisation.
 
Description Substantial progress was made on the six objectives of the grant:

1. The minimal requirements for DNA gating and transport by type II topoisomerases: importance for quinolone and structural studies.
We have invested considerable time and effort to develop an in vitro assay that measures the optimal arrangement of transported and gate DNA segments to ensure strand passage by topo IV. The main time consuming aspect has been the work needed to generate and validate the unique substrate required. This substrate can now be made in quantity and we are awaiting the outcome of assays with topo IV. Objective partially achieved. Ongoing.

2. Role of protein intercalation and DNA bending at the gate
We have now overexpressed and purified a large number of ParC mutants carrying specific alterations at Ile170, the residue that intercalates into the DNA helix to promote substantial bending at the DNA gate. By complementing with ParE, we have reconstituted the corresponding mutant topo IV activities and used them to probe cleavage and catalysis at the wild-type and mutant DNA gates. Although further characterisation will be necessary, these mutants show a range of activities that illuminate enzyme action at the gate. Objective partially completed.

3. Use of newly-developed protein/DNA reagents and new techniques in X-ray crystallography of topoisomerase complexes
We have developed several new tools and approaches to facilitate crystallography. First, we used fusion proteins of GyrB-GyrA and of ParE-ParC to stabilise complexes. Second, we used a second strong DNA site- the V-site, discovered by us during studies of the functional requirements for cleavage at the DNA. Third, we have been able to screen for crystals under higher than room temperature conditions. These modifications have allowed us to access high resolution structures of gyrase cleavage complexes and 3-gate structures of topo IV (see sections 5 and 6 below), two areas that had proven difficult heretofore. Original objective met.
Our work has been published:
Arnoldi E, Pan X-S and Fisher LM (2013) Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases. Nucleic Acids Res 41, 9411-9423

4. Irreversible antibiotic trapping of the gate: selective reaction with clerocidin
A number of different approaches varying the DNA substrate have been tried in efforts to increase the resolution of topo IV complexes with clerocidin, an antibiotic with an unusual mode of action. Despite concerted efforts, thus far, we have yet to obtain co-crystals with a resolution greater than 4.5Å. Work ongoing.

5. Focus on DNA gyrase: basis of selective quinolone targeting
By using fusion proteins comprised of GyrB and GyrA subunits, we have succeeded in a major goal of producing high resolution (3.2 to 3.6Å) structures of pneumococcal gyrase with DNA and three different quinolones. These structures finally allow comparison topo IV versus gyrase complexes for the same bacterial species providing insight on selective targeting. This work is currently being written up for publication. Original objective met.

6. Higher order structures from X-ray crystallography, EM and SAXS
We have solved the first crystal structures of an 'open clamp' 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. One complex involving E-site DNA stabilised with levofloxacin on topo IV was solved at 6.5Å (PDB ID: 4JUO): a second structure containing V-site DNA and no drug was solved at 3.7Å (PDB ID: 4I3H), revealing a bent DNA gate and a second linear DNA helix at a previously unreported site. This second DNA represents the first structure of a gate-DNA before association and bending at the gate. The ATPase domains comprising the enzyme N-gate are folded back and linked to TOPRIM domains on the same side of the enzyme, two features that ensure access for DNA and cleavage enhancing drugs. The structure is a significant advance in understanding the catalytic cycle of type II topoisomerases and has key impications for rational drug design. Original objective met.
The work has been published:
Laponogov I, Veselkov DA, Crevel IMT, Pan XS, Fisher LM and Sanderson (2013) Structure of an 'open' clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport. Nucleic Acids Res. 41, 9911-9923. (Highlighted as a paper of special interest and importance by F1000 (Faculty of 1000).
(We have not pursued EM or SAXS approaches as work on gyrase has recently appeared (Papillon et al (2013) Nucleic Acids Res 41, 7815-7827) and is at far lower resolution (23 Å) than given by our X-ray structures).

We have also solved the high resolution crystal structures of quinolone cleavage complexes formed with a gate DNA and two novel highly potent 7,8 bridged fluoroquinolones developed by Achaogen, a California-based biotech company. The drugs are interesting because the 7,8 ring fusion constrains motion of the drug enhancing potency and potentially provides a platform for further modifications that may optimize tight binding to the drug pocket on topo IV. The structures have been uploaded on to the PDB (4KPE and 4KPF) and the work has been published:
Laponogov I, Pan XS, Veselkov DA, Cirz RT, Wagman A, Moser HE, Fisher LM, Sanderson MR (2016) Exploring the active site of the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex with novel 7,8-bridged fluoroquinolones. Open Biology 6: pii: 160157.

Additional work (not in the original proposal):

We have overexpressed and purified the ATPase domain of topo IV enabling structural and mechanistic work. We successfully crystallised a closed clamp form of the ATPase gate complete with a trapped transport DNA segment threaded through the ATPase hole, the first visualisation of this key intermediate (deposited as PDB 5J5Q). The trapped DNA is kinked and carries an intercalated nucleoside triphosphate at the kink. Along with a second ATPase-DNA structure (with DNA bound on the side of the protein -PDB 5J5P), the results show how a type II topoisomerase recognises, and captures the transported DNA, a major advance. The work has been published:
Laponogov I, Pan XS, Veselkov DA, Skamrova GB, Umrekar TR, Fisher LM and Sanderson MR (2018) Trapping of the transport-segment DNA by the ATPase domains of a type II topoisomerase. Nature Communications 9: 2579. (Recommended as a 'milestone paper' by the Faculty of 1000).

The studies described above relate to the S. pneumoniae enzymes. Using recombinant means, we have also expressed a number of gyrase and topo IV proteins from Gram-negative pathogens that will allow comparison with the Gram-positive S. pneumoniae system. To aid such analysis, we have solved a crystal structure of a quinolone-DNA cleavage complex of Klebsiella pneumoniae topo IV (PDB entry 4PXP). Work has been published-
Veselkov DA, Laponogov I, Pan XS, Selvararajah J, Skamrova GB, Branstrom A, Narasimhan J, Prasad JVNV, Fisher LM and Sanderson MR (2016) Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex. Acta Cryst D72: 488-496. doi: 10.1107/S20159798316001212
Exploitation Route Our open access high resolution structures of the open clamp state of topoisomerase II has allowed us to propose a plausible mechanism for the enzyme catalytic cycle that illuminates aspects of how enzymes act as molecular machines. The full structures will be useful in understanding binding by drugs that engage multiple domains of the enzyme including the ATPase domains. Indeed, we have already had contacts from companies with inhibitors acting in this novel manner.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Our work revealing the disposition of all three gates of the bacterial topoisomerase II complex provide the highest resolution structures to date of the full protein, a key target for clinically important quinolones and for the development of new agents. Our structures have been published and the mechanistic importance is being recognised through citation. Moreover, our interactions with pharma indicate substantial interest and likely extensive use in guiding drug design.
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic
 
Description Technology transfer 
Organisation Novartis Institutes for BioMedical Research (NIBR)
Country United States 
Sector Private 
PI Contribution Transfer of techniques and expertise
Collaborator Contribution Provision of materials
Impact No outcomes thus far
Start Year 2014
 
Description Topoisomerase inhibitors active against fluoroquinolone-resistant gram-negative bacteria 
Organisation Novartis Institutes for BioMedical Research (NIBR)
Country United States 
Sector Private 
PI Contribution Joint collaboration involving Dr Mark Sanderson (Kings' College London), my group and Novartis. Was a technology transfer arrangement. We produced enzymes and DNA substrates and crystallized the proteins with a drug provided and solved the X-ray structures. The successful structural work on topo IV-drug cleavage complexes used the same Klebsiella pneumoniae topo IV protein constructs and followed the same crystallization protocol that we had published in 2016 (Veselkov DA, Laponogov I, Pan XS, Selvarajah J, Skamrova GB, Branstrom A, Narasimhan J, Vara Prasad JVN, Fisher LM and Sanderson MR (2016) Structure of a quinolone-stabilised cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex. Acta Cryst D72, 488-496 (structure on issue cover)(work supported by BBSRC grants BB/H00405/X1 and BB/K10069/1 to L Mark Fisher). Following subsequent effort over several years, the work was written up and published in 2020 (see below in outcomes). The work is directly relevant to the current MRC MR/T000848/1 grant and both Fisher and Sanderson contributed to the writing and submission of the paper.
Collaborator Contribution The project involved a substantial amount of innovative chemical synthesis, microbiology, structure-function analysis, biochemistry and toxicology complemented by our structural and biochemical input and expertise.
Impact Identification and optimisation of novel 4-(aminomethyl)quinolin-2(1H)-ones inhibitors of gyrase/topo IV designed to be active against fluoroquinolone-resistant gram-negative bacteria. The approach was multidisciplinary involving synthetic chemistry, structural biology, biochemistry, microbiology and toxicology approaches. The work has been published- Skepper CK, Armstrong D, Balibar CJ, Bauer D, Bellamacina C, Benton BM, Bussiere D, De Pascale G, De Vicente J, Dean CR, Dhumale B, Fisher LM, Fuller J, Fulsunder M, Holder LM, Hu C, Kantariya B, Lapointe G, Leeds JA, Li X, Lu P, Lvov A, Ma S, Madhavan S, Malekar S, McKenney D, Mergo W, Metzger L, Moser HE, Mutnick D, Noeske J, Osborne C, Patel A, Patel D, Patel T, Prajapati K, Prosen KR, Reck F, Richie JL, Rico A, Sanderson MR, Satasia S, Sawyer WS, Selvarajah J, Shah N, Shanghavi K, Shu W, Thompson KV, Traebert M, Vala A, Vala L, Veselkov DA, Vo J, Wang M, Widya M, Williams SL, Xu Y, Yue Q, Zang R, Zhou B, Rivkin A (2020) Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria. J Med Chem 63: 7773-7816. PMID: 32634310 DOI: 10.1021/acs.jmedchem.0c00347.
Start Year 2020
 
Description Topoisomerase inhibitors active against fluoroquinolone-resistant gram-positive bacteria 
Organisation Novartis Institutes for BioMedical Research (NIBR)
Department Infectious Diseases
Country United States 
Sector Private 
PI Contribution Joint collaboration involving Dr Mark Sanderson (Kings' College London), my group and Novartis. Was a technology transfer arrangement. We produced enzymes and DNA substrates and crystallized the proteins with a drug provided and solved the X-ray structures. The successful structural work on topo IV-drug cleavage complexes used the same Klebsiella pneumoniae topo IV protein constructs and followed the same crystallization protocol that we had published in 2016 (Veselkov DA, Laponogov I, Pan XS, Selvarajah J, Skamrova GB, Branstrom A, Narasimhan J, Vara Prasad JVN, Fisher LM and Sanderson MR (2016) Structure of a quinolone-stabilised cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex. Acta Cryst D72, 488-496 (structure on issue cover)(work supported by BBSRC grants BB/H00405/X1 and BB/K10069/1 to L Mark Fisher). Following subsequent effort over several years, the work was written up and published in 2020 (see below in outcomes). The work is directly relevant to the current MRC MR/T000848/1 grant and both Fisher and Sanderson contributed to the writing and submission of the paper.
Collaborator Contribution The project involved a substantial amount of innovative chemical synthesis, microbiology, structure-function analysis, biochemistry and toxicology complemented by our structural and biochemical input and expertise.
Impact Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria. Lapointe G, Skepper CK, Holder LM, Armstrong D, Bellamacina C, Blais J, Bussiere D, Bian J, Cepura C, Chan H, Dean CR, De Pascale G, Dhumale B, Fisher LM, Fulsunder M, Kantariya B, Kim J, King S, Kossy L, Kulkarni U, Lakshman J, Leeds JA, Ling X, Lvov A, Ma S, Malekar S, McKenney D, Mergo W, Metzger L, Mhaske K, Moser HE, Mostafavi M, Namballa S, Noeske J, Osborne C, Patel A, Patel D, Patel T, Piechon P, Polyakov V, Prajapati K, Prosen KR, Reck F, Richie DL, Sanderson MR, Satasia S, Savani B, Selvarajah J, Sethuraman V, Shu W, Tashiro K, Thompson KV, Vaarla K, Vala L, Veselkov DA, Vo J, Vora B, Wagner T, Wedel L, Williams SL, Yendluri S, Yue Q, Yifru A, Zhang Y, Rivkin A.J Med Chem. 2021 May 13;64(9):6329-6357. doi: 10.1021/acs.jmedchem.1c00375. Epub 2021 Apr 30.PMID: 33929852
Start Year 2021