Assay heterogeneity and IC50 determination

The included studies used various assays to determine IC50 values for NDM-1 inhibition. Methodological variability in substrate selection, detection methods, and reaction conditions was observed across studies. These differences influence inhibitory potency and limit the reliability of direct numerical comparisons across studies. Therefore, cross-study comparisons were performed conservatively and treated as qualitative unless the same assay formats were used. To address this variability, the included studies were classified according to the primary assay used to determine IC50 values to support within-group comparability, as shown in Table 1.

Study group | Study | Primary IC50 assay | Secondary validation assay(orthogonal, selectivity, safety) | IC50readout | Methodological robustness of IC50 reporting | Limitation
Group A | [41] | Fluorogenic kinetic hydrolysis (fluorocillin) | MIC determination (broth microdilution, K. pneumoniae E. coli); FICI synergy analysis; cytotoxicity assay (CCK-8 assay, LDH release, in vivo acute toxicity in mice) | Fluorescence kinetics (RFU/time) | High | ACE activity is not assessed; Limited mechanistic depth
[42] | Fluorogenic kinetic hydrolysis (fluorocillin) | Spectrophotometric imipenem hydrolysis assay and Ki determination; ACE selectivity; MIC potentiation assays (imipenem +tested compound); Determination of binding pose via molecular docking and dynamic studies | Fluorescence kinetics(RFU/time) | High | Human cell viability and cytotoxicity are not assessed
Group B | [43] | Spectrophotometric assay of meropenem with initial rate IC50 determination | MIC potentiation assays (meropenem +tested compound); Prediction of binding pose via solution state NMR and molecular docking studies | absorbance at 360 nm (meropenem hydrolysis) | High | ACE activity is not assessed; Human cell viability and cytotoxicity are not assessed
[38] | Spectrophotometric assay of imipenem with initial rate IC50 determination | MIC potentiation assays (meropenem +tested compound) | High
Group C | [39] | Kinetic-based inhibition assay* | MIC determination (broth microdilution, E. coli); X-ray crystallography (NDM-1); ACE activity is assessed | Not mentioned clearly* | High | Limited mechanistic depth, primary IC50 assay is not mentioned clearly*
[40] | Prescreen (inhibition, % at 1 mM) → dose response IC50 determination | MIC determination (broth microdilution, K. pneumoniae E. coli); X-ray crystallography (BcII/IMP-1/VIM-2 captopril complexes not NDM-1) | Not reported in the context** | Low | Assay conditions not fully reported; limited mechanistic depth; no orthogonal validation; ACE activity is not assessed; human cell viability and cytotoxicity are not assessed

Group A comprised fluorogenic kinetic hydrolysis assays, whereas group B included studies using spectrophotometric hydrolysis assays. Group C encompassed two studies that did not fit into either approach.

Methodological robustness was further evaluated based on the presence of supporting evidence beyond the primary assay. Orthogonal validation refers to functional assays that confirm IC50 using different experimental formats such as minimum inhibitory concentration (MIC) assays, antibiotic potentiation assays, FICI analysis, or kinetic inhibition assays. Safety evaluation refers to cytotoxicity and in vivo toxicity testing.

Selectivity assays are experiments used to assess activity against angiotensin-converting enzymes (ACE), helping determine whether compounds retain specificity for NDM-1 while minimizing potential off-target effects associated with the captopril scaffold. These assays are important for evaluating therapeutic safety and distinguishing optimized derivatives from non-selective inhibitors. Structural and biophysical characterization involves X-ray crystallography and NMR, which determine the binding interactions between the inhibitor and NDM-1.

Robustness was considered high when the IC50 was supported by at least one orthogonal secondary assay confirming inhibitor activity in a different assay format. A Moderate level of robustness was assigned when secondary evidence was present, without orthogonal validation to confirm the results from the primary IC50 assay. However, the studies may still include additional safety, selectivity, and structural assays, such as X-ray crystallography, molecular docking, or dynamics studies. A low level of robustness was assigned when conclusions relied primarily on the primary IC50 essay, with no secondary evidence. Notably, structural or computational data alone, without orthogonal validation, were insufficient to achieve high robustness, as they did not directly confirm inhibitory activity.