1432. Exploring Cell Wall Targets to Overcome Mycobacterium tuberculosis (Mtb): Ceftriaxone (CRO) Inhibits LdtMt2, a Major Peptidoglycan (PG) Synthase
Authors: Nguyen David C , Redmond Sarah N , Dousa Khalid M , Bethel Christopher , Taracila Magdalena A , Li Qing , Kurz Sebastian G , Pavelka Martin S , Papp-Wallace Krisztina , Holland Steven M , Kreiswirth Barry N , Boom Henry , Bonomo Robert A 
Published: December 2022

Abstract

Background
Drug-resistant tuberculosis (DR TB) is a deadly, difficult-to-treat infection, and new treatment strategies are needed. Despite the wide success of β-lactams (BLs), DR TB guidelines only include meropenem (MEM) and imipenem (IPM), given with clavulanate (CLA). BlaC, the Mtb β-lactamase, hydrolyzes CRO less efficiently than other cephems and β-lactamase inhibitors improve the in vitro susceptibility of Mtb to CRO. Surprisingly, CRO has not been evaluated in DR TB clinical studies. Moreover, the mechanisms by which CRO disrupts Mtb PG synthesis are not well characterized. CRO inhibits LdtMt1­, but activity against LdtMt2, an important Mtb PG synthase, is unknown. To explore this knowledge gap, we examined CRO inhibition of LdtMt2. In addition, we investigated if combining CRO with MEM or IPM would lower minimum inhibitory concentrations (MICs) more than each agent alone.

Methods
A panel of Mtb isolates was selected for susceptibility testing with a broth microdilution method. Timed electrospray ionization-mass spectrometry (ESI-MS) and inhibition kinetic assays were performed.

Results
CRO MICs ranged 0.25 to 16 µg/mL and lowered ≤ 0.06 to 2 µg/mL with CLA (Table 1). Fractional inhibitory concentration indices for CRO + MEM or IPM was &lt; 0.5 for six isolates, suggesting synergy. ESI-MS captured CRO-LdtMt2­ acyl-complexes at timepoints 5 to 120 min, and a 158 Da fragment loss was observed; MEM and IPM were unchanged (Table 2, Figure 1). When LdtMt2 was co-incubated with MEM and CRO together, only MEM complexes were captured. Interestingly, Kiapp­ with CRO (0.07 ± 0.01 µM) was comparable to that with MEM (0.09 ± 0.01 µM). Figure 1Mass spectrometry chromatograms with LdtMt2 (A) alone incubated with ceftriaxone at (B) 5 min, (C) 15 min, (D) 30 min, (E) 120 min. [Ldt¬Mt2] = 13.2 µM and [ceftriaxone] = 264 µM. Changes in MW are also listed in Table 1. (F) shows the structure of ceftriaxone with the proposed leaving group in red, leaving the remaining bound structure accounting for the observed change in MW.

Conclusion
CRO was effective in lowering MICs with MEM, IPM, and CLA for our Mtb isolates. Based upon ESI-MS, we found that CRO forms a stable complex with LdtMt2 and the R2 side chain is eliminated, while kinetic observations support inhibition of LdtMt2 by CRO (Figure 2). Previous work found MEM and IPM also inhibit multiple other PG synthases (e.g., PonA1, LdtMt1, LdtMt3). We hypothesize that CRO + MEM/IPM inhibits the growth of Mtb by the combined inactivation of multiple cell wall enzymes. Our observations support the further exploration of the notion of “target redundancy” as an approach to treat multidrug-resistant mycobacteria with BLs. Figure 2.Ceftriaxone-LdtMt2 adduct formation leading to release of the R2 side group

Disclosures
Krisztina Papp-Wallace, Ph.D, Merck: Grant/Research Support|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant Robert A. Bonomo, MD, NIH VA: Grant/Research Support|VenatoRx Merck Wockhardt Cystic Fibrosis Foundation: Grant/Research Support.