Antibacterial drug discovery

PROCOMCURE Biotech’s anti-bacterial drug discovery support routinely covers the testing of a novel compound under investigation for activity and efficacy against key ESKAPE pathogens – followed by determination of mutation frequency (resistance mapping), potential mode of action, and finally cytotoxicity. To this end, PCC has over the last years built up comprehensive resources – collection of standard ATCC strains (including multidrug-resistant ones) as listed; bank of clinical S. aureus and S. pneumoniae isolates; various in-house optimized assays; matching screening capabilities – to professionally assist our clients in lead identification, evaluation, profiling, prioritization, etc. Apart from a panel of regular testing options, PCC is also experienced in developing custom assays tailored to your specific requirements.

  • MIC testing
  • MBC testing
  • Synergy testing
  • Time-kill kinetics
  • Resistance profiling
  • Macromolecular analysis
  • Target identification
  • Assay development

A MIC (minimal inhibitory concentration) assay is typically performed for determining bacterial susceptibility to a novel antimicrobial compound under study (and so basically its in vitro activity). By exposing a bacterial species of choice to a serial dilution series of assayed test agent (performed in 96-well microtiter plate format in at least biological triplicates each), the lowest concentration that inhibits visible growth after overnight incubation under defined test conditions can be deciphered by measuring optical density. For further validation, any hit is then screened against a broader panel of pathogens. PROCOMCURE Biotech completes respective protocols in approximately 3 days, and offers customizable testing options (single compounds to library hits; numerous strains; standard MIC; MIC 50/80/100 determination) to accommodate laboratories of all sizes. Test compounds can be of either natural or synthetic origin.

Gram-positive pathogensGram-negative pathogens
Staphylococcus aureus (MRSA-COL; Newman; RN4220; Mu50 reference; S. epidermidis; SA113; MW2; collection of S. aureus strains overexpressing all 505 essential gene products (in progress); mutants generated in-house against most commercially available antibiotics); Streptococcus pneumoniae; Bacillus subtilis; Mycobacterium smegmatis; Lactobacillus acidophilus Acinetobacter baumannii; Enterococcus faecalis; Enterobacter aerogenes; Pasteurella multocida; Enterococcus faecium; Escherichia coli (DH5α, BL21DE3, efflux pump mutants); Haemophilus influenzae; Klebsiella pneumoniae; Pseudomonas aeruginosa (and efflux pump mutant)

The MBC (minimum bactericidal concentration) describesMIC MBC TK 1 the lowest antibacterial compound dilution which results in a 99.9% decrease from the initial starting inoculum titre, and delivers a primary impression of whether compounds act bactericidal or rather bacteriostatic. Cells from the non-turbid wells of a completed MIC assay are for this purpose plated onto agar in relation to appropriate controls, and grown colonies enumerated after further overnight incubation. Per definition, an agent is regarded bactericidal if the MBC is no more than 4x the MIC. Most infections respond just as well to bacteriostatic drugs as to bactericidal ones, although definitive killing should trigger a faster eradication and so improved clinical outcome.

Gram-positive pathogensGram-negative pathogens
Staphylococcus aureus (MRSA-COL; Newman; RN4220; Mu50 reference; S. epidermidis; SA113; MW2; collection of S. aureus strains overexpressing all 505 essential gene products (in progress); mutants generated in-house against most commercially available antibiotics); Streptococcus pneumoniae; Bacillus subtilis; Mycobacterium smegmatis; Lactobacillus acidophilus Acinetobacter baumannii; Enterococcus faecalis; Enterobacter aerogenes; Pasteurella multocida; Enterococcus faecium; Escherichia coli (DH5α, BL21DE3, efflux pump mutants); Haemophilus influenzae; Klebsiella pneumoniae; Pseudomonas aeruginosa (and efflux pump mutant)

Synergy 1Synergy testing by checkerboard titration assesses cooperative interactions of selected compound combinations. To this end, PROMCOMCURE Biotech challenges the desired pathogen with serially diluted compound concentrations bracketing the pre-determined MIC. A 90° angle rotated pipetting scheme is hereby applied to provide all possible combinations. The FIC (fractional inhibitory concentration) coefficient is finally determined all along the growth / no growth interface by dividing the MIC of the compound when used in combination by the MIC when administered alone. The FIC-index is then quantified as the sum of the 2 FIC values for each drug present in the well. Arbitrary thresholds of ≤0.5; >0.5 to ≤1.0; >1 to ≤4, and >4.0 are used to score and interprete synergistic (beneficial activity), additive (combined effect equal to the sum of the individual components), indifferent (neutral) or antagonistic (adverse interaction) effects within the paired combinations. Hints for forthcoming combinatorial treatment regimens, ideally achieving enhanced efficacy, decreased dosage and reduced side-effects whilst preventing the emergence of resistant strains, can in the long run be deduced from such in vitro data, thereby widening the spectrum of antibacterial activity.

Respective curves assess a compound‘s in vitro antimicrobialMIC MBC TK 1 activity over time. An overnight culture is diluted, incubated at 37°C for 2hrs (early exponential) or 5hrs (late exponential), challenged with any substance under study in the 1x to 10x MIC range, and plated at regular time intervals (0, 4, 6, 8, 10, 12 and 24hrs).

The percentage of dead cells is finally calculated relative to enclosed growth controls. PCC has already successfully performed a number of time-kill studies for diverse clients, following standard guidelines.

Both the increasing emergence and pandemic spreading of isolates developing multi-drug resistances to last-barrier antibiotics constitute a significant threat to global public health. Determination of spontaneous mutation events / frequencies / acquisition dynamics by either growing pathogens on agar plates supplemented with the agent under study (1x to 10x MIC range, depending on compound solubility and customer’s intention) or sequential passaging in the presence of raising compound concentrations can be used to predict the types of resistances (probably also to other antibiotics) that might arise in the clinic.

PROCOMCURE Biotech evaluates a compounds’ influence on specific macromolecular pathways (DNA, RNA, protein, cell wall, and lipid biosynthesis) by measuring the incorporation of radiolabeled precursors into logarithmic phase bacterial cultures (whole-cell labeling) above and below the pre-determined MIC value. As with all our offered services, antibiotics with known MOA serve as internal references. Antibacterial agents shown to inhibit protein synthesis can upon request be further validated using an in vitro transcription / translation assay (applying an E. coli S30 extract system). Further options are presently in development.

Following (next generation) genome sequencing of generated mutants, protein target(s) affected by a compound under study – including potential ‘off’ targets – and so underlying mechanisms of action (MOAs) might be deciphered. MOAs principally include interference with cell wall synthesis; inhibition of protein as well as DNA synthesis; disturbance of metabolic pathways; and disruption of bacterial membrane structure. Inability to generate a resistant clone might parenthetically imply that this compound has either multiple targets or exhibits a nonspecific mode of action. Again, PCC provides either single-step (direct plating of bacterial cells onto agar containing a 1x to 10x MIC compound concentration) or serial passaging protocols.

Our further capabilities – based upon extensive, multi-year research expertise – include reagent sourcing, target protein expression feasibility studies for downstream studies, (enzyme) assay development / optimization, etc., providing rapid turnaround times and consistent results (along with technology transfer as needed) for your project.


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