Peter Lewis

Amino Alcohols as Potential Antibiotic and Antifungal Leads

Publisher: MDPI AG

Date: 22-03-2022

DOI: 10.3390/MOLECULES27072050

Abstract: Five focused compound libraries (forty-nine compounds), based on prior studies in our laboratory were synthesized and screened for antibiotic and anti-fungal activity against S. aureus, E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, C. albicans and C. neoformans. Low levels of activity, at the initial screening concentration of 32 μg/mL, were noted with analogues of (Z)-2-(3,4-dichlorophenyl)-3-phenylacrylonitriles which made up the first two focused libraries produced. The most promising analogues possessing additional substituents on the terminal aromatic ring of the synthesised acrylonitriles. Modifications of the terminal aromatic moiety were explored through epoxide installation flowed by flow chemistry mediated ring opening aminolysis with discreet sets of amines to the corresponding amino alcohols. Three new focused libraries were developed from substituted anilines, cyclic amines, and phenyl linked heterocyclic amines. The aniline-based compounds were inactive against the bacterial and fungal lines screened. The introduction of a cyclic, such as piperidine, piperazine, or morpholine, showed % inhibition when evaluated at 32 μg/mL compound concentration against methicillin-resistant Staphylococcus aureus. Examination of the terminal aromatic substituent via oxirane aminolysis allowed for the synthesis of three new focused libraries of afforded amino alcohols. Aromatic substituted piperidine or piperazine switched library activity from antibacterial to anti-fungal activity with ((Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(4-methylpiperazin-1-yl)propoxy)phenyl)acrylonitrile), ((Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(4-(4-hydroxyphenyl)piperazin-1-yl)propoxy)-phenyl)acrylonitrile) and ((Z)-3-(4-(3-(4-cyclohexylpiperazin-1-yl)-2-hydroxypropoxy)-phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile) showing % inhibition of Cryptococcus neoformans var. grubii H99 growth at 32 μg/mL. While (Z)-3-(4-(3-(cyclohexylamino)-2-hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile, (S,Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)acrylonitrile, (R,Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(piperidin-1-yl)propoxy)phenyl)acrylonitrile, (Z)-2-(3,4-dichlorophenyl)-3-(4-(2-hydroxy-3-(D-11-piperidin-1-yl)propoxy)phenyl)-acrylonitrile, and (Z)-3-(4-(3-(4-cyclohexylpiperazin-1-yl)-2-hydroxypropoxy)-phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile 32 μg/mL against Staphylococcus aureus.

RNA polymerases from low G+C gram-positive bacteria

Publisher: Informa UK Limited

Date: 08-08-2021

DOI: 10.1080/21541264.2021.1964328

Molecular Basis for RNA Polymerase-Dependent Transcription Complex Recycling By The Helicase-like Motor Protein HelD.

Publisher: Research Square Platform LLC

Date: 16-07-2020

DOI: 10.21203/RS.3.RS-40738/V1

Abstract: In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using cryo-electron microscopy and single-particle analysis, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the extraordinary conformational changes that occur in RNAP driven by HelD interaction. HelD has a unique 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a dramatic conformational change in the primary channel leading to removal and recycling of the stalled polymerase.

Protein‐protein interactions as antibiotic targets: A medicinal chemistry perspective

Publisher: Wiley

Date: 13-07-2018

DOI: 10.1002/MED.21519

Abstract: There are 27 small molecule protein-protein interaction (PPI) modulators in Phase I, II, and III clinical trials targeting cancer, viruses, autoimmune disorders, and as immune suppression agents. Targeting PPIs as an antibiotic drug discovery strategy remains in relative infancy by comparison. However, a number of molecules are in development which target PPI within the replisome, isome, transcriptome, and translatome are showing significant promise at the medicinal chemistry stage of drug development. Hence, the success of future PPI agents as antibiotics will build upon the techniques and design strategies of these molecules.

Structural characterisation of the complete cycle of sliding clamp loading inEscherichia coli

Publisher: Cold Spring Harbor Laboratory

Date: 21-07-2023

DOI: 10.1101/2023.07.20.549978

Abstract: Ring-shaped DNA sliding cl s are essential for DNA replication and genome maintenance. Cl s need to be opened or trapped open and chaperoned onto DNA by cl loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place cl s around DNA remains limited. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed cl on and off a primer-template DNA that represent all intermediates in the cl loading process. We show that the ATP-bound CLC first binds to a cl , then constricts to hold onto it. The CLC then expands to open the cl with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing ATP hydrolysis and cl closing around DNA. Although both yeast and E. coli CLCs open cl s by crab claw-like motions, they do it by the CLC expanding in opposite directions. These structures provide critical high-resolution snapshots of cl loading by the E. coli CLC, revealing how the molecular machine works.

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Publisher: Cold Spring Harbor Laboratory

Date: 19-08-2021

DOI: 10.1101/2021.08.18.456904

Abstract: Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor protein distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work we have undertaken the first comprehensive investigation of the phylogenetic ersity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. Therefore, HelD proteins represent an important new class of transcription factor associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

Publisher: Springer Science and Business Media LLC

Date: 18-12-2020

DOI: 10.1038/S41467-020-20157-5

Abstract: In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

Tuning the Anthranilamide Peptidomimetic Design to Selectively Target Planktonic Bacteria and Biofilm

Publisher: MDPI AG

Date: 15-03-2023

DOI: 10.3390/ANTIBIOTICS12030585

Abstract: There is a pressing need to develop new antimicrobials to help combat the increase in antibiotic resistance that is occurring worldwide. In the current research, short hiphilic antibacterial and antibiofilm agents were produced by tuning the hydrophobic and cationic groups of anthranilamide peptidomimetics. The attachment of a lysine cationic group at the tail position increased activity against E. coli by -fold (from μM to 15.6 μM) and greatly reduced cytotoxicity against mammalian cells (from ≤20 μM to ≥150 μM). These compounds showed significant disruption of preformed biofilms of S. aureus at micromolar concentrations.

RNA polymerases from Low G+C Gram Positive Bacteria

Publisher: Cold Spring Harbor Laboratory

Date: 07-06-2021

DOI: 10.1101/2021.06.06.447298

Abstract: The low G+C Gram positive bacteria represent some of the most medically and industrially important microorganisms. They are relied on for the production of food and dietary supplements, enzymes and antibiotics, as well as being responsible for the majority of nosocomial infections and serving as a reservoir for antibiotic resistance. Control of gene expression in this group is more highly studied than in any bacteria other than the Gram negative model Escherichia coli , yet until recently no structural information on RNA polymerase (RNAP) from this group was available. This review will summarise recent reports on the high resolution structure of RNAP from the model low G+C representative Bacillus subtilis , including the role of auxiliary subunits δ and ε, and outline approaches for the development of antimicrobials to target RNAP from this group.

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Publisher: Wiley

Date: 12-2021

DOI: 10.1002/MBO3.1251

Abstract: Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long‐lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP‐dependent motor proteins distantly related to helicases. It was first identified in the model Gram‐positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram‐positive bacteria. In this work, we have undertaken the first comprehensive investigation of the phylogenetic ersity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram‐negative bacteria. In summary, HelD proteins represent an important new class of transcription factors associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Mechanism of transcription modulation by the transcription-repair coupling factor

Publisher: Oxford University Press (OUP)

Date: 31-05-2022

DOI: 10.1093/NAR/GKAC449

Abstract: Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

University Of Oxford

Location: United Kingdom of Great Britain and Northern Ireland

View Organisation

The University Of Newcastle

Location: Australia

View Organisation

Hunter Biological Solutions

Location: Australia

View Organisation

Discovery Projects - Grant ID: DP0664370

Start Date: 2006

End Date: 12-2010

Amount: $273,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882295

Start Date: 2008

End Date: 06-2009

Amount: $225,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0449482

Start Date: 2004

End Date: 07-2007

Amount: $210,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100047

Start Date: 2016

End Date: 09-2016

Amount: $380,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP210100365

Start Date: 02-2021

End Date: 02-2024

Amount: $692,195.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0561089

Start Date: 2005

End Date: 12-2010

Amount: $25,064.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989861

Start Date: 2009

End Date: 12-2010

Amount: $650,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0211014

Start Date: 2001

End Date: 12-2002

Amount: $337,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110100190

Start Date: 2011

End Date: 12-2017

Amount: $300,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100127

Start Date: 2016

End Date: 12-2016

Amount: $355,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0561173

Start Date: 2005

End Date: 12-2005

Amount: $207,189.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989105

Start Date: 2009

End Date: 11-2009

Amount: $495,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100078

Start Date: 02-2011

End Date: 12-2011

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0453608

Start Date: 2004

End Date: 12-2004

Amount: $579,230.00

Funder: Australian Research Council