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DNA topoisomerase inhibitor

Authors|

Elizabeth Michalczyk, Kay Hommernick, Iraj Behroz, Marcel Kulike, Zuzanna Pakosz-Stępień, Lukasz Mazurek, Maria Seidel, Maria Kunert, Karine Santos, Holger von Moeller, Bernhard Loll, John B. Weston, Andi Mainz, Jonathan G. Heddle, Roderich D. Süssmuth & Dmitry Ghilarov

Typesetter|Dorothy

Keywords

albicidin, DNA topoisomerase inhibitor, dual binding mechanism, cryo-EM structure 2

Background

Currently, there’s an urgent need for a new class effective against Gram-negative bacteria. This is the background of this research.

Gram-negative bacteria originating in hospitals cause a high death toll, and are becoming more pan-resistant. However, there hasn’t been a new class discovered targeting Gram-negative bacteria in the last 50 years, and there has been high attrition rates of drug candidates in preclinical trials, meaning that challenges from Gram-negative bacteria reduces the strength of these candidates.

The combination of DNA intercalation and dimer interface

There currently exist many candidates and in-use antibiotics, and they often share a common principle. The bacterial type II topoisomerase DNA gyrase, along with the homologous topoisomerase IV (Topo IV) are essential for bacteria but absent in humans, making them main targets in antibiotics. The DNA gyrase is made up of two GyrA and two GyrB monomers. It negatively supercoils DNA: this uses a temporary double-strand break in the bound segment (G or gate segment) and the adjacent (T or transported) segment of the same DNA is guided through the G segment, in a process that consumes ATP.

Current possible solutions for Gram-negative bacteria include the following. First, the F1 gyrase poisoning mechanism uses intercalating into cleaved DNA to form the FQ-protein-DNA complex, leading to cell death. However, this option has severe side-effects, so is currently restricted use by a black box warning in the US. Second is using ‘novel bacterial topoisomerase inhibitors’ (NBTIs), such as gepotidacin, to mimic the effective process of FQs. This is currently under phase III trials, and though NBTIs usually are cardiotoxic, gepotidacin itself only has a mild effect on heart rate. The final option is the one under investigation: albicidin. It’s produced by Xanthomonas albilineans, a bacteria that causes leaf scald disease in sugarcane. It inhibits DNA gyrase at nanomolar concentration, and has derivatives with higher effectivity and good safety profile. This albicidin is made up of six residues: methyl p-coumaric acid (MCA1), p-aminobenzoic acid (pABA2 and pABA4), β-cyano-L-alanine (Cya3) and 4-amino-2-hydroxy-3-methoxybenzoic acid (pMBA5 and pMBA6).



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Experiments

To start with, the team tested for requirements of the stabilization of the cleavage complex by albicidin. DNA gyrase uses gyrase cleavage core fusion, which is a truncated and fused structure. It’s made up of C-terminal topoisomerase-primase (TOPRIM) domain of GyrB monomer and N-terminal part of GyrA monomer. From previous experience, the team hypothesized that the albicidin binding requires a DNA strand passage event. Because the stabilization of gyrase cleavage complex by microcin (an antibiotic peptide) B17 requires a long DNA segment, they expected the same stabilization requirement to be true for the stabilization of the gyrase cleavage complex, and tested a range of different DNA fragments with strong gyrase-binding sites (SGSs) of phage Mu. They found that strong cleavage of the 217 bp Mu SGS fragment (Mu217) occurs in the presence of albicidin and ATP or the analogue ADPNP, which could not be hydrolysed. Also, they made the important discovery that almost no cleavage was detected in the presence of less than 150 bp DNA strand or in the absence of nucleotide, meaning that the presence of a long DNA fragment, which can bind to GyrA CTD was essential for cleavage. Further, the presence of the nucleotide and the length of the DNA substrate collectively enabled the cleavage complex stabilization. Since Mu217 was able to be cleaved, they scaled up production of the fragment for use of structure determination in the following stages.

Following that, the team determined the specific mechanism through which albicidin traps DNA gyrase. The gyrase-DNA-albicidin complex with the ATP homologue ADPNP exhibited high structural homogeneity and virtually no DNA static disorder. This enabled the structure of the cleavage core to be determined at local resolution of 2.6Å. The clarity of the data was a significant improvement from previous ones. The CTD of GyrA with wrapped DNA and the ATPase belonging to GyrB were also seen, though at lower resolution due to intrinsic flexibility of structure, and so were not modeled. The overall structure of the complex resembles that between gyrase and the aforementioned NBTI gepotidacin, with almost perfect symmetry except the DNA and cleavage of the DNA fragment at the site expected.



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Result

First, the N-terminal of albicidin intercalates, that is to say, inserts between the two strands of the DNA. The N-terminal (MCA1) insertion was at the cleave site, 5’-T/GATTT-3’, the exact cleave site for FQ, though with differing mechanisms, while the terminal hydroxyl of the MCA reached the opposite strand at the corresponding C/A. Second, at the opposite end, the C-terminal, similarly inserted between the α3 and α3’, two opposing helices. This allowed the formation of the GyrA/GyrA’ interface. Third, there’s significant sized shifts at the GyrA/GyrA’ interface, characterized by a sliding door motion. Similar significant shifts were also present in GyrB and at the DNA ends. The result was that the catalytic intermediate state resembles a state between being partially open and fully open. Fourth, the aforementioned N-terminal of albicidin, only occupied half of the cleavage site (also called the TG pocket). This led to pronounced distortion of DNA in the symmetrically-related AA pocket, enabling strong selectivity for the TG pocket. Fifth, the pMBA5 and pMBA6 of albicidin bound in a pseudosymmetric manner, occupying the hydrophobic pockets in GyrA and GyrA’. Sixth, they used the photocrosslinkable (able to form a photo-induced covalent bond) analogue of albicidin to determine the binding mode of albicidin. They found that gyrase and the catalytic conditions were both needed for the N-terminus of albicidin to interact with cleaved DNA. Seventh, the mechanism of the metal-ion dependent DNA cleavage by the Topo IV counterpart–type II topoisomerase–was determined. Here, the ion was largely ascertained to be Mg2+. The activity of metal-ion dependent DNA cleavage may be as follows: the B configuration was used to store the metal temporarily during the strand passage event, then the magnesium ion remained attached to residues Asp500 throughout. Though the recruitment of a second ion before DNA relegation (joining of DNA fragments after breakage), which this process occurs, is also possible.

Moreover, the potentiated albicidin derivatives were used to show binding heterogeneity. First, Albi-1 is a derivative that exhibits higher pharmacological characteristics and nanomolar-range activity toward FQ-resistant pathogens. Initially, analysis of this derivative showed three stages coexisting in the structure. This was possibly due to the shorter N-terminal of Albi-1, which allowed both TG and AA pocket occupation. Second, the C-terminally truncated derivative, where pMBA6 was removed, shows a decrease in activity, suggesting that both pMBA5 and pMBA6 were important for trapping gyrase. Third, they tested the Albi-2 and Albi-3 derivatives, where MCA1 was replaced by equivalents that were also able to intercalate in DNA. For Albi-2, there’s a four time better activity in terms of gyrase inhibition compared to Albi-3.

Besides this, the site directed mutagenesis also confirmed the previously determined binding mode of albicidin. The team used multiple sequence alignment of GyrA subunits provided by the ConSurf server to prevent artificially designed mutations from creating unnatural substitutions. They found the following through examining the resulting variants and the original albicidin: First, binding was almost completely unaffected by mutations in the quinolone-resistance determining region (QRDR) of GyrA. Second, though the Lys447 mutation in GyrB QRDR offered some resistance, especially to the parent compound, its effect could be successfully overcome by the alterations of the DNA-intercalating moiety as demonstrated by Albi-3. Third, the GyrAA67Q mutation provided some resistance to Albi-1 or Albi-3, but it was not likely to naturally occur because it disrupted the gyrase function.

Finally, the target specificity of albicidin is determined. The team initially suspects a dual-target of Topo IV and gyrase. Albicidin derivatives can all stabilize cleavage with Topo IV, except Albi-2, though Albi-1 has best performance. Albi-1 and Albi-3 for inhibition require only 1/10 the concentration of the rest of the derivatives. In any case, albicidin can inhibit Topo IV, though with lower efficiency than with gyrase, but further modifications can increase the efficiency.

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Discussion

As a conclusion, this research provides structure-based design that preserves albicidin’s distinct and potent inhibition mechanism. Albi-1 and Albi-3 show safety and efficacy in animal models. The α3/α3’ pocket is unique and doesn’t overlap with other classes of inhibitors. Albi-1 and Albi-3 have nanomolar activity in cleavage complex stabilization, which is more potent than NBTIs, as well as a high proportion of cleaved complexes. Albicidin targets Topo IV and gyrase, making resistance harder to develop. Activity of current derivatives is lower, but can be improved. In the future, for successful clinical application of albicidins, solubility, plasma stability, and plasma-protein binding need to be increased, while monitoring the toxicity to eukaryotic topoisomerase II.

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The significance to our life

Protease is an important macromolecular substance in living organisms. The peptide antibiotics discussed in this paper are of great significance in clinical treatment. The research on the mechanism of its inhibition of DNA gyrase is very important for the current and future applications of this antibiotic, are closely related to our lives.

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Reference


Michalczyk, Elizabeth, et al.“抗菌肽白霉素致拓扑异构酶中毒的分子机制。”自然催化(2023):1-16。

https://www.nature.com/articles/s41929-022-00904-1#citeas


Michalczyk, Elizabeth, et al. "Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin." Nature Catalysis (2023): 1-16. https://www.nature.com/articles/s41929-022-00904-1#citeas



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