Ribonucleotide reductase, a novel drug target for gonorrhea
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This paper is of interest to biochemists and those focused on development of novel antibiotics. The authors present two small molecules that specifically target the essential ribonucleotide reductase of the causative agent of gonorrhea, with biochemical, biophysical, and biological data supporting the efficacy of these molecules both in vitro and in mouse models. Overall, this is a comprehensive study providing some interesting insights to guide the development of new therapies for malaria.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
Abstract
Antibiotic-resistant Neisseria gonorrhoeae (Ng ) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α 4 β 4 state in the presence of the β subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.
Article activity feed
-
-
Author Response:
Reviewer #1:
In this study, the authors seek to understand the target and mechanism of action of two structurally related orally available antibiotic drug candidates active against Neisseria gonorrhoeae (Ng). The experimental approach involves a detailed investigation of drug efficacy in bacterial culture experiments and a mouse model for gonorrhea infections, along with biochemical experiments to identify the drug target. The latter experiments include discovery of resistance-inducing mutations in class Ia ribonucleotide reductase (RNR), in vitro validation of the ability of the Ng inhibitors to diminish enzyme activity, and structural studies to evaluate the effects of the compounds on Ng RNR structure. The work succeeds in providing convincing evidence for inhibition of the RNR but it does not fully explain how …
Author Response:
Reviewer #1:
In this study, the authors seek to understand the target and mechanism of action of two structurally related orally available antibiotic drug candidates active against Neisseria gonorrhoeae (Ng). The experimental approach involves a detailed investigation of drug efficacy in bacterial culture experiments and a mouse model for gonorrhea infections, along with biochemical experiments to identify the drug target. The latter experiments include discovery of resistance-inducing mutations in class Ia ribonucleotide reductase (RNR), in vitro validation of the ability of the Ng inhibitors to diminish enzyme activity, and structural studies to evaluate the effects of the compounds on Ng RNR structure. The work succeeds in providing convincing evidence for inhibition of the RNR but it does not fully explain how the drug candidates bind to the enzyme. Although the findings represent an important advance that could motivate other work in exploiting bacterial RNRs for antibiotic drug development, conclusions about the mechanism of action could be better supported by more thorough understanding of inhibitor-enzyme interaction. This insight would be important for improving drug design and broad expansion of the approach to other pathogens. Additionally, the inhibitors are billed as narrow-spectrum antibiotic candidates, but these claims are based on analysis of a small and specialized group of bacteria that are not likely to contain or exclusively rely on a class Ia RNR. It is not clear from this study if the inhibitors could affect growth of commensal organisms that contain aerobic RNRs.
Table S7 has been included to highlight the known forms of RNRs present in various organisms. The selective inhibition of Neisseria gonorrhoeae and N. meningitidis is corroborated by the presence of only a single RNR, type Ia. Other aerobic Gram-negative organisms, including E. coli and K. pneumoniae, have type Ia, Ib and III, and are not inhibited by the PTC compounds. The presence or multiple forms of RNR types may be the explanation for other organisms lacking sensitivity to the PTC compounds, but the data are not conclusive. Obtaining conclusive data will require additional experimentation with engineered knock-out isogenic strains to fully elucidate why other organisms are not inhibited.
Reviewer #2:
This study by Narasimhan et al. describes the identification of ribonucleotide reductase (RNR), a critical enzyme in all organisms, as a new target for treatment of antibiotic-resistant gonorrhea, via a novel mechanism for RNR inhibition. The authors begin with the identification of two inhibitors that selectively target Neisseria gonorrhoeae, including multidrug resistant strains, over other pathogens and microbiota. They then show that these inhibitors target the synthesis of DNA, but not by the mechanism of other members of this class of compounds; instead, isolation of resistant mutants indicates that the class Ia ribonucleotide reductase of this organism is the target of the molecules. These results are supported by in vitro activity assays of the RNR, along with electron microscopy characterization of the RNR, showing that the resistance mutations disrupt the protein's ability to form a ring-like inhibited state, implying that the mechanism of action of the compounds involves that ring-like state. While other compounds have been shown to induce formation of the inhibited state of the human RNR, the mechanism of inhibition of the gonorrheal RNR evidenced here is distinct. Finally, the authors present data from a mouse infection model showing efficacy of the compounds. The comprehensive nature of this study, from small molecule to in vitro analysis to in vivo efficacy, is compelling, and the results are of interest both from an enzymological perspective and from the development of new strategies to combat important pathogens. There are two issues that I believe the authors should address to support two important aspects of their work, the mechanisms of inhibition and resistance:
- The major question that sticks in my mind after reading this manuscript is the mechanism by which the molecules inhibit the RNR. They act as potent inhibitors in vitro, and the identification of the resistance mutations, H25R and S41L, which interfere with the ability of the RNR to form the inactive a4b4 form, are strong pieces of evidence in favor of the authors' proposal that the inhibitors "potentiate conversion of its active a2b2 state to an inactive a4b4." The clincher of this argument would be EM evidence that the presence of the inhibitors leads to a4b4 formation, just as the H25R and S41L variants do, or (perhaps simpler) size exclusion chromatography or direct evidence from another analytical method pointing to formation of the a4b4 species.
Relatedly, it would also be helpful if the dependency of inhibition on dATP would be clarified. Figure S6 suggests that dATP is not required for this state, but on p. 14, line 12, the authors write "the dependency on a dATP-induced inactive a4b4 state also explains why the Ng and Ec Ia RNRs are both sensitive to these inhibitors…" and on p. 20, line 27, it is also implied that dATP could be involved. Please clarify this point.
We have carried out additional experiments to probe the mechanism of inhibition of PTC compounds, and our results suggest that there is likely to be more than one mode of enzyme inhibition. One mode of inhibition does appear to be related to dATP-inhibition and thus α4β4 ring formation. In particular, we now present data that show that PTC-672 and PTC-846 potentiate the inhibitory effects of dATP (Figure 4). However, the PTC compounds themselves are not dATP mimics; they do not appear to be able to substantially increase the amount of α4β4 ring formation in vitro. This finding is based on new mass photometry data that we now present (Supplemental data Figure S8, S9). We also now present data that show that PTC-672 can inhibit (to some degree) variants of Ng RNR that can’t form rings (H25L and S41L) (Figure S7), providing evidence of a second mode of inhibition. Collectively, these results indicate that the mode(s) of inhibition of PTC compounds are complex. We don’t know why E. coli is not less effected than Ng by these compounds. It could be that the class Ia RNR in E. coli is equally inhibited but that the presence of multiple RNRs in E. coli is protective. It is possible that the synergistic effect of the PTC compounds with dATP is less dramatic for the E. coli enzyme or that the second (unknown) mode of inhibition is not in play in E. coli. Much more work will need to be done to answer this question and that work is out of the scope of this paper. We are pleased, however, to present several new pieces of data, including the mass photometry results, to fill out this story.
- The observation that the resistance mutant strains have lower fitness is an interesting and important one. I suggest that the authors determine whether this decreased fitness might be the result of the mutations in the RNR leading to lower activity - the authors should give the activities of the H25R and S41L with the normal substrate, vs. wild-type alpha. If the activities are similar to wild-type, perhaps the authors could suggest another potential explanation. One that seems possible to me is that the loss of dATP inhibition (see Fig S5) might lead to loss of fitness via misregulation of (deoxy)nucleotide pools.
H25R and S41L Ng RNR variants are impaired in their ability to be down-regulated by dATP but are not otherwise impaired. Our hypothesis is as the reviewer suggests that loss of fitness is due to the inability to down-regulate RNR, which misregulates nucleotide pools. This phenomenon was observed for E. coli RNR class Ia. In the case of E. coli class Ia RNR, S39F and E42K variants were shown only to be impaired in their ability to be down-regulated by dATP, and yet, these mutations were linked to a mutator phenotype (see Chen 2018 JBC 293, 10404 and Ahluwalia 2012 DNA Repair 11, 480). Not being able to turn off an RNR is problematic for the cell.
-
Evaluation Summary:
This paper is of interest to biochemists and those focused on development of novel antibiotics. The authors present two small molecules that specifically target the essential ribonucleotide reductase of the causative agent of gonorrhea, with biochemical, biophysical, and biological data supporting the efficacy of these molecules both in vitro and in mouse models. Overall, this is a comprehensive study providing some interesting insights to guide the development of new therapies for malaria.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
In this study, the authors seek to understand the target and mechanism of action of two structurally related orally available antibiotic drug candidates active against Neisseria gonorrhoeae (Ng). The experimental approach involves a detailed investigation of drug efficacy in bacterial culture experiments and a mouse model for gonorrhea infections, along with biochemical experiments to identify the drug target. The latter experiments include discovery of resistance-inducing mutations in class Ia ribonucleotide reductase (RNR), in vitro validation of the ability of the Ng inhibitors to diminish enzyme activity, and structural studies to evaluate the effects of the compounds on Ng RNR structure. The work succeeds in providing convincing evidence for inhibition of the RNR but it does not fully explain how the …
Reviewer #1 (Public Review):
In this study, the authors seek to understand the target and mechanism of action of two structurally related orally available antibiotic drug candidates active against Neisseria gonorrhoeae (Ng). The experimental approach involves a detailed investigation of drug efficacy in bacterial culture experiments and a mouse model for gonorrhea infections, along with biochemical experiments to identify the drug target. The latter experiments include discovery of resistance-inducing mutations in class Ia ribonucleotide reductase (RNR), in vitro validation of the ability of the Ng inhibitors to diminish enzyme activity, and structural studies to evaluate the effects of the compounds on Ng RNR structure. The work succeeds in providing convincing evidence for inhibition of the RNR but it does not fully explain how the drug candidates bind to the enzyme. Although the findings represent an important advance that could motivate other work in exploiting bacterial RNRs for antibiotic drug development, conclusions about the mechanism of action could be better supported by more thorough understanding of inhibitor-enzyme interaction. This insight would be important for improving drug design and broad expansion of the approach to other pathogens. Additionally, the inhibitors are billed as narrow-spectrum antibiotic candidates, but these claims are based on analysis of a small and specialized group of bacteria that are not likely to contain or exclusively rely on a class Ia RNR. It is not clear from this study if the inhibitors could affect growth of commensal organisms that contain aerobic RNRs.
-
Reviewer #2 (Public Review):
This study by Narasimhan et al. describes the identification of ribonucleotide reductase (RNR), a critical enzyme in all organisms, as a new target for treatment of antibiotic-resistant gonorrhea, via a novel mechanism for RNR inhibition. The authors begin with the identification of two inhibitors that selectively target Neisseria gonorrhoeae, including multidrug resistant strains, over other pathogens and microbiota. They then show that these inhibitors target the synthesis of DNA, but not by the mechanism of other members of this class of compounds; instead, isolation of resistant mutants indicates that the class Ia ribonucleotide reductase of this organism is the target of the molecules. These results are supported by in vitro activity assays of the RNR, along with electron microscopy characterization of …
Reviewer #2 (Public Review):
This study by Narasimhan et al. describes the identification of ribonucleotide reductase (RNR), a critical enzyme in all organisms, as a new target for treatment of antibiotic-resistant gonorrhea, via a novel mechanism for RNR inhibition. The authors begin with the identification of two inhibitors that selectively target Neisseria gonorrhoeae, including multidrug resistant strains, over other pathogens and microbiota. They then show that these inhibitors target the synthesis of DNA, but not by the mechanism of other members of this class of compounds; instead, isolation of resistant mutants indicates that the class Ia ribonucleotide reductase of this organism is the target of the molecules. These results are supported by in vitro activity assays of the RNR, along with electron microscopy characterization of the RNR, showing that the resistance mutations disrupt the protein's ability to form a ring-like inhibited state, implying that the mechanism of action of the compounds involves that ring-like state. While other compounds have been shown to induce formation of the inhibited state of the human RNR, the mechanism of inhibition of the gonorrheal RNR evidenced here is distinct. Finally, the authors present data from a mouse infection model showing efficacy of the compounds. The comprehensive nature of this study, from small molecule to in vitro analysis to in vivo efficacy, is compelling, and the results are of interest both from an enzymological perspective and from the development of new strategies to combat important pathogens. There are two issues that I believe the authors should address to support two important aspects of their work, the mechanisms of inhibition and resistance:
- The major question that sticks in my mind after reading this manuscript is the mechanism by which the molecules inhibit the RNR. They act as potent inhibitors in vitro, and the identification of the resistance mutations, H25R and S41L, which interfere with the ability of the RNR to form the inactive a4b4 form, are strong pieces of evidence in favor of the authors' proposal that the inhibitors "potentiate conversion of its active a2b2 state to an inactive a4b4." The clincher of this argument would be EM evidence that the presence of the inhibitors leads to a4b4 formation, just as the H25R and S41L variants do, or (perhaps simpler) size exclusion chromatography or direct evidence from another analytical method pointing to formation of the a4b4 species.
Relatedly, it would also be helpful if the dependency of inhibition on dATP would be clarified. Figure S6 suggests that dATP is not required for this state, but on p. 14, line 12, the authors write "the dependency on a dATP-induced inactive a4b4 state also explains why the Ng and Ec Ia RNRs are both sensitive to these inhibitors..." and on p. 20, line 27, it is also implied that dATP could be involved. Please clarify this point.
- The observation that the resistance mutant strains have lower fitness is an interesting and important one. I suggest that the authors determine whether this decreased fitness might be the result of the mutations in the RNR leading to lower activity - the authors should give the activities of the H25R and S41L with the normal substrate, vs. wild-type alpha. If the activities are similar to wild-type, perhaps the authors could suggest another potential explanation. One that seems possible to me is that the loss of dATP inhibition (see Fig S5) might lead to loss of fitness via misregulation of (deoxy)nucleotide pools.
-
-