Selective binding of small molecules to Vibrio cholerae DsbA offers a starting point for the design of novel antibacterials

Oligoribonuclease (Orn), a member of the DEDDh superfamily, can hydrolyse 2–5 nt nanoRNAs to mononucleotides. It is involved in maintaining the intracellular levels of RNA, c-di-GMP signalling and transcription initiation in many bacterial species. Here, the crystal structure of Orn from Vibrio cholerae O1 El Tor (VcOrn) is reported at a resolution of 1.7 Å. VcOrn, which consists of nine α-helices and six β-strands, crystallizes with a single monomer in the asymmetric unit but forms a homodimer via crystallographic twofold symmetry. Electron density is observed in the active pocket that corresponds to an intersubunit N-terminal expression tag with sequence GPLGSHHH. The positively charged N-terminal tag binds in the negatively charged nucleotide-binding pocket with a buried surface area of ∼500 Å2. The N-terminal tag interacts with VcOrn via π–π stacking with two conserved residues involved in nucleotide binding, as well as via salt bridges and hydrogen bonds. The structure reported here reveals that the active pocket can accommodate polypeptides in addition to nucleotides, thus providing an important starting point for investigation into substrate modification and inhibitor design targeting VcOrn.

Download Full-text

Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae

Microbiology ◽

10.1099/mic.0.079319-0 ◽

2014 ◽

Vol 160 (9) ◽

pp. 1855-1866 ◽

Cited By ~ 11

Author(s):

Shreya Dasgupta ◽

Pallabi Basu ◽

Ritesh Ranjan Pal ◽

Satyabrata Bag ◽

Rupak K. Bhadra

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Small Molecules ◽

Vibrio Cholerae ◽

Stringent Response ◽

Synthetase Activity ◽

Gram Negative Bacteria ◽

Amino Acid Residues ◽

Gram Negative

In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3′-diphosphate 5′-triphosphate (pppGpp) and guanosine 3′,5′-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp0 cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.

Download Full-text

Automated Design of Macrocycles for Therapeutic Applications: from Small Molecules to Peptides and Proteins

10.26434/chemrxiv.12640781 ◽

2020 ◽

Author(s):

Daniel Sindhikara ◽

Michael Wagner ◽

Paraskevi Gkeka ◽

Stefan Guessregen ◽

Garima Tiwari ◽

...

Keyword(s):

Small Molecules ◽

Cyclic Peptides ◽

Conformational Stability ◽

Three Dimensional ◽

Cell Permeability ◽

Linear Molecule ◽

Retrospective Case ◽

Therapeutic Modalities ◽

Protein Interfaces ◽

Starting Point

<div>Macrocycles and cyclic peptides are increasingly attractive therapeutic modalities as they often have </div><div>improved affinity, are able to bind to extended protein interfaces and otherwise have favorable </div><div>properties. Macrocyclization of a known binder molecule has the potential to stabilize its bioactive </div><div>conformation, improve its metabolic stability, cell permeability and in certain cases oral </div><div>bioavailability. Herein, we present an in silico approach that automatically generates, evaluates and </div><div>proposes cyclizations utilizing a library of well-established chemical reactions and reagents. Using the </div><div>three-dimensional (3D) conformation of the linear molecule in complex with a target protein as </div><div>starting point, this approach identifies attachment points, generates linkers, evaluates the </div><div>conformational landscape of suitable linkers and their geometric compatibility and ranks the resulting </div><div>molecules with respect to their predicted conformational stability and interactions with the target </div><div>protein. As we show here with several prospective and retrospective case studies, this procedure can </div><div>be applied for the macrocyclization of small molecules and peptides and even PROTACs and proteins.</div><div>The presented approach is an important step towards the enhanced utilization of macrocycles and</div><div>cyclic peptides as attractive therapeutic modalities.</div>

Download Full-text

Identification of 6-(piperazin-1-yl)-1,3,5-triazine as a chemical scaffold with broad anti-schistosomal activities

Wellcome Open Research ◽

10.12688/wellcomeopenres.16069.1 ◽

2020 ◽

Vol 5 ◽

pp. 169

Author(s):

Gilda Padalino ◽

Iain W. Chalmers ◽

Andrea Brancale ◽

Karl F. Hoffmann

Keyword(s):

Virtual Screening ◽

Small Molecules ◽

Adult Worm ◽

In Silico ◽

Egg Production ◽

Homology Modelling ◽

Homology Model ◽

Biological Data ◽

Significant Activity ◽

Starting Point

Background: Schistosomiasis, caused by infection with blood fluke schistosomes, is a neglected tropical disease of considerable importance in resource-poor communities throughout the developing world. In the absence of an immunoprophylactic vaccine and due to over-reliance on a single chemotherapy (praziquantel), schistosomiasis control is at risk should drug insensitive schistosomes develop. In this context, application of in silico virtual screening on validated schistosome targets has proven successful in the identification of novel small molecules with anti-schistosomal activity. Methods: Focusing on the Schistosoma mansoni histone methylation machinery, we herein have used RNA interference (RNAi), ELISA-mediated detection of H3K4 methylation, homology modelling and in silico virtual screening to identify a small collection of small molecules for anti-schistosomal testing. A combination of low to high-throughput whole organism assays were subsequently used to assess these compounds’ activities on miracidia to sporocyst transformation, schistosomula phenotype/motility metrics and adult worm motility/oviposition readouts. Results: RNAi-mediated knockdown of smp_138030/smmll-1 (encoding a histone methyltransferase, HMT) in adult worms (~60%) reduced parasite motility and egg production. Moreover, in silico docking of compounds into Smp_138030/SmMLL-1’s homology model highlighted competitive substrate pocket inhibitors, some of which demonstrated significant activity on miracidia, schistosomula and adult worm lifecycle stages together with variable effects on HepG2 cells. Particularly, the effect of compounds containing a 6-(piperazin-1-yl)-1,3,5-triazine core on adult schistosomes recapitulated the results of the smp_138030/smmll-1 RNAi screens. Conclusions: The biological data and the structure-activity relationship presented in this study define the 6-(piperazin-1-yl)-1,3,5-triazine core as a promising starting point in ongoing efforts to develop new urgently needed schistosomicides.

Download Full-text

Selective binding of small molecules to Vibrio cholerae DsbA offers a starting point for the design of novel antibacterials

10.33774/chemrxiv-2021-stw44 ◽

2021 ◽

Author(s):

Geqing Wang ◽

Biswaranjan Mohanty ◽

Martin Williams ◽

Bradley Doak ◽

Rabeb Dhouib ◽

...

Keyword(s):

Vibrio Cholerae ◽

Crystal Structures ◽

Small Molecule ◽

Sodium Taurocholate ◽

Structural Basis ◽

Human Pathogens ◽

X Ray ◽

X Ray Crystallography ◽

Hydrophobic Groove ◽

Starting Point

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446 ± 10 µM. The same benzimidazole compound has ~8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD > 3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveals the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

Download Full-text

Identification of 6-(piperazin-1-yl)-1,3,5-triazine as a chemical scaffold with broad anti-schistosomal activities

Wellcome Open Research ◽

10.12688/wellcomeopenres.16069.2 ◽

2020 ◽

Vol 5 ◽

pp. 169

Author(s):

Gilda Padalino ◽

Iain W. Chalmers ◽

Andrea Brancale ◽

Karl F. Hoffmann

Keyword(s):

Virtual Screening ◽

Small Molecules ◽

Adult Worm ◽

In Silico ◽

Egg Production ◽

Homology Modelling ◽

Homology Model ◽

Biological Data ◽

Significant Activity ◽

Starting Point

Background: Schistosomiasis, caused by infection with blood fluke schistosomes, is a neglected tropical disease of considerable importance in resource-poor communities throughout the developing world. In the absence of an immunoprophylactic vaccine and due to over-reliance on a single chemotherapy (praziquantel), schistosomiasis control is at risk should drug insensitive schistosomes develop. In this context, application of in silico virtual screening on validated schistosome targets has proven successful in the identification of novel small molecules with anti-schistosomal activity. Methods: Focusing on the Schistosoma mansoni histone methylation machinery, we herein have used RNA interference (RNAi), ELISA-mediated detection of H3K4 methylation, homology modelling and in silico virtual screening to identify a small collection of small molecules for anti-schistosomal testing. A combination of low to high-throughput whole organism assays were subsequently used to assess these compounds’ activities on miracidia to sporocyst transformation, schistosomula phenotype/motility metrics and adult worm motility/oviposition readouts. Results: RNAi-mediated knockdown of smp_138030/smmll-1 (encoding a histone methyltransferase, HMT) in adult worms (~60%) reduced parasite motility and egg production. Moreover, in silico docking of compounds into Smp_138030/SmMLL-1’s homology model highlighted competitive substrate pocket inhibitors, some of which demonstrated significant activity on miracidia, schistosomula and adult worm lifecycle stages together with variable effects on HepG2 cells. Particularly, the effect of compounds containing a 6-(piperazin-1-yl)-1,3,5-triazine core on adult schistosomes recapitulated the results of the smp_138030/smmll-1 RNAi screens. Conclusions: The biological data and the structure-activity relationship presented in this study define the 6-(piperazin-1-yl)-1,3,5-triazine core as a promising starting point in ongoing efforts to develop new urgently needed schistosomicides.

Download Full-text

Extensive Crystallographic Fragment-Based Approach to Design SARS CoV2 3CLpro Main Protease Inhibitors and Related Metadata

10.26434/chemrxiv.13550246.v1 ◽

2021 ◽

Author(s):

Sarfraz Ahmad ◽

Iskandar Abdullah ◽

Yean Kee Lee ◽

Mamoona Nazir ◽

Muhammad Usman Mirza ◽

...

Keyword(s):

Small Molecules ◽

Md Simulation ◽

Data Bank ◽

Binding Pocket ◽

New Drugs ◽

Main Protease ◽

Binding Data ◽

Starting Point ◽

Bona Fide ◽

Further Development

<p>3CLpro is a vital protein for the SARS-CoV-2 replications and its inhibition using small molecules is a <i>bona fide</i> approach used to develop new drugs against the virus. In this study, a comprehensive crystallography-guided fragment-based drug discovery approach was employed to design new inhibitors for SARS-CoV-2 3CLpro. Protein Data Bank was explored to find small molecules cocrystallized with SARS-CoV-2 3CLpro. The fragments sitting in the binding pocket (87) were interactively coupled using various linkers with the intention to get molecules having the same orientation as those of the constituting fragments. In total, 1251 couples were prepared and converted to maximum possible stereoisomers using LigPrep for screening using Glide (standard precision and extra precision), AutoDock Vina, and Prime MMGBSA. Top 22 hits having conformations similar to their cocrystallized fragments were selected for MD simulation on Desmond. MD simulation suggested that 15 hits had conformations very close to their constituting fragments. Results indicated that these hits were computationally reliable and could be considered for further development. This suggests that the study could provide a benchmark starting point for the further design of SARS-CoV-2 3CLpro inhibitors with improved binding (data provided). <br></p>

Download Full-text

An in vitro and in silico identification of antibiofilm small molecules from seawater metaclone SWMC166 against Vibrio cholerae O1

Molecular and Cellular Probes ◽

10.1016/j.mcp.2018.03.003 ◽

2018 ◽

Vol 39 ◽

pp. 14-24 ◽

Cited By ~ 3

Author(s):

Murugan Rajalaxmi ◽

Rajamohamed Beema Shafreen ◽

Karuppiah Chithiraiselvi ◽

Shunmugiah Karutha Pandian

Keyword(s):

Small Molecules ◽

Vibrio Cholerae ◽

In Silico ◽

Vibrio Cholerae O1 ◽

In Silico Identification

Download Full-text

Identification of 4-aminoquinoline core for the design of new cholinesterase inhibitors

PeerJ ◽

10.7717/peerj.2140 ◽

2016 ◽

Vol 4 ◽

pp. e2140 ◽

Cited By ~ 11

Author(s):

Yao Chen ◽

Yaoyao Bian ◽

Yuan Sun ◽

Chen Kang ◽

Sheng Yu ◽

...

Keyword(s):

Small Molecules ◽

Cholinesterase Inhibitor ◽

Cholinesterase Inhibitors ◽

Simple Structure ◽

Therapeutic Strategies ◽

Inhibitory Effects ◽

Inhibitory Potency ◽

Ache Inhibitors ◽

Starting Point ◽

Derivatives Of

Inhibition of acetylcholinesterase (AChE) using small molecules is still one of the most successful therapeutic strategies in the treatment of Alzheimer’s disease (AD). Previously we reported compound T5369186 with a core of quinolone as a new cholinesterase inhibitor. In the present study, in order to identify new cores for the designing of AChE inhibitors, we screened different derivatives of this core with the aim to identify the best core as the starting point for further optimization. Based on the results, we confirmed that only 4-aminoquinoline (compound 04 and 07) had cholinesterase inhibitory effects. Considering the simple structure and high inhibitory potency against AChE, 4-aminoquinoline provides a good starting core for further designing novel multifunctional AChEIs.

Download Full-text