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NNadir

(33,368 posts)
Wed Sep 23, 2020, 08:36 PM Sep 2020

A Convergent Synthesis Scheme for Overcoming Virginiamycin Antibiotic Resistance.

The paper I'll discuss in this post is this one: Synthetic group A streptogramin antibiotics that overcome Vat resistance (Seipel et al., Nature (2020). https://doi.org/10.1038/s41586-020-2761-3)

Now that we are experiencing a global pandemic, we are more familiar with the state of affairs that existed for most of human history; deadly infectious agents routinely killed people in huge numbers, with the result that life expectancy was close to 1/2 of what it is today. My grandmother for instance, died in her early 40's from a bacterial infection - my mother was 11 years old - that today might have easily been cured with penicillin, or any other of a number of antibiotics in our arsenal.

However, since pathogenic bacteria have short doubling times, with many generations passing in a single day, there is ample opportunity for them to rapidly evolve resistance to those drugs, thus making them ultimately useless.

There is a big economic problem with anti-infectious medications and that is that they cure diseases. This is very different than is the case with, say, a blood pressure medicine that manages but doesn't cure the disease. The innovator company can collect sales for as long as they can keep its patent life going. Our economic system, given the extremely high cost and extremely risky nature of investing in the discovery of new drugs, does not select for curative agents (unless the disease is so widespread that sales will be enormous even if people are cured).

Worse from an economic standpoint is that if a drug can address a resistant strain that has evolved resistance to existing common antibiotics - well trained doctors will not write scrips for it unless all other medications have failed. This is responsible medicine: It follows that a drug that cures diseases that cannot be cured by "ordinary" means, will not sell.

When I was a kid, I briefly managed a combinatorial chemistry lab for a company that claimed - as marketing - that it was making tools that would enable the discovery of fifty drugs per year. The chemical libraries that I and my team made for this effort, under the direction of the chief scientific officer, didn't develop any drugs. Part way along in this effort, I figured out why this was inevitable, but nobody wanted to hear what I had to say, and I quit and found another job. (No, it was not as bad as Theranos; they were not making stuff up. It was just that the scientific assumptions did not hold experimental water.)

The paper I've cited above is kind of "chemical library" - like, a kind of better steered combinatorial chemistry. Don't give up the ship as they say. It is far superior in focus to what my company was doing 30 odd years ago.

From the introduction:

Natural product antibiotics often have poor characteristics as therapeutic agents1 and are subject to resistance mechanisms that have arisen through coevolution4. A primary method to improve natural antibiotics for human use is semisynthesis—that is, chemical modification of natural products obtained by biological production. This method has improved the pharmacological properties of many natural product classes but has achieved only limited success in overcoming resistance mechanisms1. Recently, advances in chemistry have enabled access to several antibiotic classes by fully synthetic routes that provide renewed methods to overcome resistance5,6.

Streptogramin antibiotics comprise two structurally distinct groups (A and B)3 (Extended Data Fig. 1a) that act synergistically to achieve bactericidal activity in many organisms7 by inhibiting the bacterial ribosome8. Group A antibiotics bind to the peptidyl transferase centre (PTC) and increase affinity for the group B component in the adjacent nascent peptide exit tunnel9. Resistance to the A component mediates high-level resistance to the combination, whereas resistance to the B component results in intermediate resistance10. Similar to other antibiotics that target the PTC, resistance to group A streptogramins can be mediated by the ATP-binding cassette F (ABC-F) family proteins that dislodge antibiotics11 or by Cfr methylases that methylate A2503 of the 23S rRNA to sterically block binding12. A specific resistance mechanism for group A streptogramins is deactivation by virginiamycin acetyltransferases (Vats)2. These proteins acetylate the C14 alcohol, resulting in steric interference and disruption of a crucial hydrogen bond. The combination of vat(A) and vgb(A) genes (which deactivate the B component) is the most clinically relevant streptogramin-resistance genotype in S. aureus in France, where streptogramins (under the trade name Pyostacine) are used orally for skin and soft tissue infections13,14 as well as bone and joint infections15. Semisynthesis has improved water solubility (for example, Synercid16) and increased potency (for example, NXL-10317), but methods to overcome resistance to the class have yet to be discovered. Fully synthetic routes to group A streptogramins have been previously developed18,19,20,21,22,23,24,25,26,27,28, but these routes have not been applied to the synthesis of new analogues. Here we report optimization of our initially reported route18 and its application to the synthesis of analogues designed to overcome streptogramin resistance.


Streptogramins are antibiotics whose carbons are arranged (on a molecular scale of course) in large rings, rings much larger than the 5, 6, 7 and 8 member rings that are fairly common in chemistry.

The basic structure is suggested by this combinatorial chemistry synthetic scheme, Figure 1:



The caption:

a, Convergent route to VM2 from seven building blocks. b, Eighteen group A streptogramins accessed by building block variation. The fragments displayed in the dashed boxes represent the structural variability compared to the parent scaffold (VM2). Overall yields for the synthesis of each analogue for the left half sequence (top number) and for the right half sequence (bottom number) are displayed. **Instead of a ketone, madumycin II (34) contains the following substitution at C16: ?-H, ?-OH. c, Access to 34 analogues (17 in each diastereomeric series) with C3 side-chain variability by means of carbamate formation followed by desilylation. d, Synthesis of tertiary-amine-containing analogues by oxidation and reductive amination. e, C16-fluorinated analogues. DCC, N,N?-dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; HATU, hexafluorophosphate azabenzotriazole tetramethyl uronium; OTBS, O-(tert-butyldimethylsilyl)hydroxylamine.


In antibiotic talk, "MIC" is called the "minimum inhibitory concentration," the lower the number, the better the antibiotic:



The caption:

a, MIC values for selected analogues against an expanded panel of pathogens. Each MIC was obtained in technical triplicate. The bars to the right display in vitro translation that occurs in the presence of 10 ?M of each analogue (relative to dimethylsulfoxide (DMSO)). b, MIC values against clinical isolates of S. aureus with Vat resistance genes. Ranges of values obtained from technical replicates are displayed; asterisks indicate MIC values that were obtained in technical triplicate and biological triplicate. c, A mouse thigh model of infection with S. aureus CIP 111304 (n = 5 biologically independent animals per group, with the exception of the 2 h infection control where n = 4 per group, examined over one experiment). Each mouse is individually plotted, the centre line is the mean, and the upper and lower whiskers bound the standard deviation from the mean. For detailed statistical analysis, see Extended Data Table 3. CFU, colony-forming units.


Years ago, the testing of potential drugs from chemical libraries basically depended on ligand binding assays. Thing have gotten far more sophisticated and now relies on actual measurement of protein-drug interactions:

In agreement with their low MIC values, 4 (half-maximal inhibitory concentration (IC50) of 40 ± 10 nM (mean ± s.d.)) and 47 (IC50 of 70 ± 20 nM) inhibited translation more effectively than VM2 (IC50 of 500 ± 200 nM) in vitro (Fig. 3a). The similar IC50 values of 4 and 47 suggest that their MIC differences are due to factors other than improved ribosome inhibition. To quantify deactivation by VatA, we measured rates of C14 acetylation using purified VatA for 4 and 47. The approximately 2.5-fold reduction in catalytic efficiency (kcat/Km) does not linearly account for the 8- to 16-fold reduction in MIC values in the plasmid-mediated VatA S. aureus strain (strain 2) (Fig. 2a–c), but it is similar to the reduction in the MIC values of the clinical isolates of S. aureus (strains 13–19). Nonlinear correlation of MIC value to drug deactivation can result from the contribution of other factors such as cellular accumulation, other resistance mechanisms, and efflux39,40. To determine the structural contributions of 47 to a low rate of VatA acetylation, we obtained an X-ray co-crystal structure (Fig. 3b), which reveals displacement of Leu110 by 1.5 Å compared to VM1 in VatA (PDB code 4HUS2) due to steric clash with the C4 extension of 47.

To explore the structural basis for antimicrobial activity, we characterized several analogues bound to the E. coli ribosome using cryo-EM (Fig. 3c, d, Extended Data Figs. 5, 6). The PTC is highly conserved across pathogenic species of bacteria, and the E. coli ribosome is an appropriate model for group A streptogramin binding in both Gram-negative and Gram-positive organisms9. The 2.6-Å structure of analogue 47 bound to the ribosome clearly reveals the position of the C4-allyl extension, which projects towards the streptogramin B binding site and makes contacts with A2062, U2585 and U2586 (Extended Data Fig. 5). This extension also adopts a less strained conformation when ribosome-bound than when VatA-bound (calculated ?2.3 kcal mol?1) (Extended Data Fig. 7, Extended Data Table 2). This difference, along with protein conformational changes (Fig. 3b), could contribute to the observed differences in acetylation rates between 4 and 47. In the presence of VS1, the C4 extension adopts a strained conformation similar to its conformation in VatA but is probably stabilized by hydrophobic interactions with the B component (Fig. 3d).

Ligand strain may also have a role in the efficacy of 46. Predicted low-energy conformations of 46 position the arylcarbamate extension directly over the macrocycle (Extended Data Fig. 7); however, the structures of 46 bound to the ribosome in the presence or absence of VS1 (Fig. 3b, Extended Data Fig. 5) showed density for the extension in the P-site. The isoquinoline portion of the extension sits between A2602 and C2452, without making specific contacts with either. The proximity of C29 to U2585 may explain the difference in activity between the two diastereomeric series at this position (40a-q and 41a-q) (Fig. 1c).


A cartoon about the binding:



The caption:

a, Summary of VatA acetylation kinetics and in vitro inhibition of the E. coli ribosome by 4 and 47. Error bars denote s.d. (3 technical replicates). For detailed statistical analysis, see Extended Data Table 3. b, X-ray crystal structures of VM1 bound to VatA (PDB code 4HUS; 2.4 Å ) and 47 bound to VatA at 3.2-Å resolution. Distances shown are measured between carbons of the C4 extension of 47 and Leu110 in the VM1-bound VatA structure (in orange dashes, 2.1 Å ) and in the 47-bound VatA structure (in marine dashes, 3.6 Å ). c, 2.7-Å cryo-EM Coulomb potential density map (contoured in dark blue at 4.0? and light grey at 1.0? ) for ribosomes bound to 46 and VS1. d, 2.8-Å cryo-EM Coulomb potential density map for ribosomes bound to 47 and VS1. e, Overlay of selected PTC-site antibiotics shows how the side chain of 46 and the extension of 47 occupy areas distinct to previously characterized antibiotics. f, Overlay of P-site tRNA (dark grey, PDB code 1VY4) with the cryo-EM structure of ribosome-bound 46 reveals that the side chain extends into the P-site and mimics the terminal adenosine (A2450) of the tRNA.


Despite all this nonsense in the government, a true Confederacy of Dunces, to steal a line from John Kennedy Toole, scientists are still working to save our pathetic little butts from things other than Covid.

This is a cute little paper (actually a very, very good paper) that evokes a certain sense of nostalgia in me.

Life is interesting as hell, and then you die.

Have a nice day tomorrow.
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