Structure-guided microbial targeting of antistaphylococcal prodrugs

Carboxy ester prodrugs have been widely employed as a means to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can successfully mask problematic chemical features that prevent cellular uptake and can be used to target delivery of compounds to specific tissues. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, curbing their potential therapeutic applications. While carboxy ester-based prodrug targeting is feasible, it has been limited in microbes due to a paucity of information about the selectivity of microbial esterases. Here we identify the bacterial esterases, GloB and FrmB, that are required for carboxy ester prodrug activation in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, identifying several promoieties likely to be serum esterase-resistant while still being microbially labile. These studies lay the groundwork for structure guided design of antistaphyloccal promoieties, enabling a massive expansion of the antistaphyloccal druggable space.

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Structure-guided microbial targeting of antistaphylococcal prodrugs

eLife ◽

10.7554/elife.66657 ◽

2021 ◽

Vol 10 ◽

Author(s):

Justin J Miller ◽

Ishaan T Shah ◽

Jayda Hatten ◽

Yasaman Barekatain ◽

Elizabeth A Mueller ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cellular Uptake ◽

Oral Absorption ◽

Therapeutic Potential ◽

Structural Basis ◽

Substrate Specificities ◽

Ester Substrate ◽

Human And Mouse ◽

Ester Prodrugs ◽

Microbial Esterase

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of anti-staphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens.

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Antimicrobial prodrug activation by the staphylococcal glyoxalase GloB

10.1101/2020.07.23.214460 ◽

2020 ◽

Author(s):

Marwa O. Mikati ◽

Justin J. Miller ◽

Damon M. Osbourn ◽

Naomi Ghebremichael ◽

Ishaan T. Shah ◽

...

Keyword(s):

Oral Absorption ◽

Loss Of Function ◽

Prodrug Activation ◽

Glyoxalase Ii ◽

Human Sera ◽

Cellular Permeability ◽

Ester Prodrug ◽

Ester Prodrugs

ABSTRACTWith the rising prevalence of multidrug-resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g. serum half-life, oral absorption, solubility, toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating “prodrugs” that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, S. schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologs are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species-or genus-level prodrug targeting strategies.

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Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

Communications Biology ◽

10.1038/s42003-021-02357-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Sushant Kumar ◽

Arunabh Athreya ◽

Ashutosh Gulati ◽

Rahul Mony Nair ◽

Ithayaraja Mahendran ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pathogenic Bacteria ◽

Major Facilitator Superfamily ◽

Single Domain ◽

Fluoroquinolone Resistance ◽

Structural Basis ◽

Antibody Complex ◽

Complementarity Determining Regions ◽

Major Facilitator ◽

Alternating Access

AbstractTransporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

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Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome

mBio ◽

10.1128/mbio.00395-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 14

Author(s):

Matthew J. Belousoff ◽

Zohar Eyal ◽

Mazdak Radjainia ◽

Tofayel Ahmed ◽

Rebecca S. Bamert ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Structural Information ◽

Structural Basis ◽

Common Mechanism ◽

Resistance Mutations ◽

Antimicrobial Drugs ◽

Linezolid Resistance ◽

Drug Modification ◽

Antibiotic Resistance Mutations ◽

Shed Light

ABSTRACT An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

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Structural basis for different substrate specificities of two ADP-ribose pyrophosphatase

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767308091630 ◽

2008 ◽

Vol 64 (a1) ◽

pp. C261-C261

Author(s):

T. Wakamatsu ◽

N. Nakagawa ◽

S. Kuramitsu ◽

R. Masui

Keyword(s):

Structural Basis ◽

Substrate Specificities

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Amelioration of sperm immobilisation factor-induced infertility by bacterial antigenic determinants cross-reacting with spermatozoa

Reproduction Fertility and Development ◽

10.1071/rd18300 ◽

2019 ◽

Vol 31 (3) ◽

pp. 602 ◽

Cited By ~ 4

Author(s):

Deepali Thaper ◽

Deepak K. Rahi ◽

Vijay Prabha

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Molecular Mimicry ◽

Fluorescein Isothiocyanate ◽

Antigenic Determinants ◽

Sperm Function ◽

Contraceptive Effect ◽

Normal Sperm ◽

Ameliorative Effect ◽

Human And Mouse

A strain of Staphylococcus aureus, capable of invitro immobilisation of human and mouse spermatozoa, was already present in our laboratory. Therefore, in the present study, the factor responsible (sperm immobilisation factor, SIF) was isolated and purified. It was found to compromise not only motility, but also viability, morphology and Mg2+-ATPase activity of mouse spermatozoa. Also, SIF (250μgmL−1), when administered intravaginally in female BALB/c mice before mating, showed 100% contraceptive effect. Moreover, fluorescein isothiocyanate-labelled SIF was also found to bind mouse spermatozoa and various motile as well as non-motile bacteria, indicating the presence of common SIF-binding receptors on spermatozoa and bacteria. Further, to demonstrate molecular mimicry, the amelioration of SIF-induced impairment of sperm function by a SIF-binding bacterial receptor was compelling. For this, the SIF-binding receptor from Escherichia coli (E-SBR) was purified and evaluated for its ameliorative effect on SIF-induced sperm impairment invitro and invivo. Interestingly, upon the addition of mouse spermatozoa to SIF pre-incubated with E-SBR, an ameliorative effect against SIF-induced impairment of sperm function could be observed through analysis of normal sperm parameters (motility, viability, morphology, Mg2+-dependent ATPase levels). E-SBR also blocked binding of labelled SIF to spermatozoa and bacteria and alleviated SIF-induced infertility in female BALB/c mice. This provided evidence for molecular similarities between bacteria and spermatozoa, owing to which anti-bacterial antibodies cross-reacting with spermatozoa might be produced and infertility might follow.

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Structure of an ancestral ADP-dependent kinase with fructose-6P reveals key residues for binding, catalysis, and ligand-induced conformational changes

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015376 ◽

2020 ◽

pp. jbc.RA120.015376

Author(s):

Sebastián M. Muñoz ◽

Victor Castro-Fernandez ◽

Victoria Guixe

Keyword(s):

Conformational Changes ◽

Active Site ◽

Substrate Specificities ◽

Closed Conformation ◽

Sugar Binding ◽

Bifunctional Enzymes ◽

Glucose Binding ◽

Critical Residue ◽

Key Residues ◽

Human And Mouse

ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFK), specific glucokinases (ADP-GK), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, no one exhibits fructose-6P at the active site. Employing an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with fructose-6P at its active site. Key residues for sugar-binding and catalysis were identified by alanine scanning, being D36 a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding since its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar-binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semi-closed and closed).

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