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. 2024 Mar 22;29(7):1431.
doi: 10.3390/molecules29071431.

Camel-Derived Nanobodies as Potent Inhibitors of New Delhi Metallo-β-Lactamase-1 Enzyme

Rahma Ben Abderrazek  1 , Emna Hamdi  1   2 , Alessandra Piccirilli  2 , Sayda Dhaouadi  1 , Serge Muyldermans  3 , Mariagrazia Perilli  2 , Balkiss Bouhaouala-Zahar  1   4
Affiliations

Camel-Derived Nanobodies as Potent Inhibitors of New Delhi Metallo-β-Lactamase-1 Enzyme

Rahma Ben Abderrazek et al. Molecules. .

Abstract

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of β-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting β-lactamases might become essential to reduce and prevent bacterial infections. Nanobodies (Nbs), the smallest antigen-binding single-domain fragments derived from Camelidae heavy-chain-only antibodies, targeting enzymes, are innovative alternatives to develop effective inhibitors. The biopanning of an immune VHH library after phage display has helped to retrieve recombinant antibody fragments with high inhibitory activity against recombinant-NDM-1 enzyme. Nb02NDM-1, Nb12NDM-1, and Nb17NDM-1 behaved as uncompetitive inhibitors against NDM-1 with Ki values in the nM range. Remarkably, IC50 values of 25.0 nM and 8.5 nM were noted for Nb02NDM-1 and Nb17NDM-1, respectively. The promising inhibition of NDM-1 by Nbs highlights their potential application in combating particular Gram-negative infections.

Keywords: NDM-1; metallo-β-lactamases; nanobodies; phage display; β-lactamase small molecule inhibitors.

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Conflict of interest statement

The authors declare not conflict of interest.

Figures

Figure 1
Figure 1
Evaluation of phage particle enrichment with target-specific Nbs during sequential biopanning steps using polyclonal phage ELISA. A total of 1010 phages from each round of panning were evaluated for their binding activity against the NDM-1 enzyme. BSA was used as a negative control. R0 represents the unpanned library, and R1–R3 represents VHH-virions eluted after rounds 1–3 of library panning. Values are the average of two replicate wells, and error bars designate the standard deviation.
Figure 2
Figure 2
Selection of NDM-1-specific VHH fragments. The periplasmic products of randomly picked clones were tested using ELISA. Clones with at least 2× stronger ELISA signals on NDM-1-coated wells vs. non-coated wells were considered positive. (A) Selection of NDM-1-specific VHH fragments after the second round of panning. (B) Selection of NDM-1-specific VHH fragments after the third round of panning.
Figure 3
Figure 3
(A) Schematic illustration of Nb in the phagemid vector pMECS. Nb sequence was tracked by a triple alanine linker, hemagglutinin (HA), and hexa-histidine (His) tags. (B) Amino acid sequences of NDM-1-specific Nbs, numbered according to IMGT [26]. CDR1, CDR2, and CDR3 are indicated in pink, blue, and purple, respectively. The Framework regions (FR) and hypervariable regions or complementarity determining regions (CDR) are indicated at the bottom of the figure.
Figure 4
Figure 4
Reactivity of purified Nbs against NDM-1. The binding ability of purified Nbs using 1 µg/mL of recombinant NDM-1 enzyme was tested using ELISA. The three Nbs showed a strong signal at least 10-fold higher compared to the background signal, to assess non-specific binding, and compared to the irrelevant control signal, to demonstrate the specificity of binding with the antigen of interest. (Error bars indicating the standard deviation represent the variability observed between biological replicates).
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