Discovery of a fragment hit compound targeting D-Ala:D-Ala ligase of bacterial peptidoglycan biosynthesis

Figures & data

Figure 1. (A) Reaction mechanism and (B) inhibitors of Ddl.

Table 1. Fragment library subsets and number of fragments used for screening on DdlB.

Fragment library subsets	Number of fragments
In-house diverse fragments	195
Purchased diverse fragments	283
Chelating fragments	214
Phosphate bioisostere fragments	159
Cycloserine analogues	31
Docked fragments to DdlB	61
Total	943

Table 2. Profiling of repurchased fragment hits from the screening.

Structure	Label	Fragment library subset	DdlB inhibition	MurA inhibition	Phosphate binding	HRP-PR assay	H2DCFDA assay	Resazurin assay	Thiol reactivity
	3	Docked fragments to DdlB	IC50 = 168 μM	64% RA at 5 mM	111% free phosphate at 5 mM	Not active	Not active	Not active	Not reactive
	4	Purchased diverse fragments	IC50 = 1790 μM	39% RA at 5 mM	29% free phosphate at 5 mM	Not active	Active	Active	Reactive
	5	Purchased diverse fragments	28% RA at 2.5 mM	6% RA at 5 mM	10% free phosphate at 5 mM	Not active	Active	Not active	Not reactive
	6	Purchased diverse fragments	25% RA at 5 mM	104% RA at 5 mM	23% free phosphate at 5 mM	Not active	Not active	Active	Not reactive
	7	Docked fragments to DdlB	47% RA at 5 mM	81% RA at 5 mM	89% free phosphate at 5 mM	Not active	Not active	Not active	Not reactive
	8	Purchased diverse fragments	95% RA at 5 mM	100% RA at 5 mM	85% free phosphate at 5 mM	Not active	Active	Not active	Not reactive

RA: Residual Activity.

Figure 2. Fragment hit 3 targeting DdlB. (A) Dose–response inhibition of DdlB for 3. (B) SPR analysis of 3 with the immobilised DdlB. 3 was injected across DdlB in serial dilutions ranging from 78.125 to 5000 µM for 40 s at a flow rate of 30 μL/min, and dissociation was followed for 40 s. Three independent measurements were performed and representative sensorgrams revealing dose-dependent binding of 3 to DdlB are shown. (C) Data were fitted to the steady-state affinity model to obtain the apparent equilibrium dissociation constant (K_D). K_D values are the mean ± standard deviation of three titrations. (D) ATP (blue sticks) and D-Ala-D-Ala (white sticks) binding sites of DdlB (PDB ID: 4C5C) with the co-crystallised ligands. (E) Computational docking of 3 to the ATP-binding site using extra-precision Glide (blue sticks) and QM-Polarized Ligand Docking (orange sticks) (PDB ID: 4C5C).

Table 3. Analogues of compound 3 that were evaluated in the DdlB inhibition assay.

Structure	Label	DdlB inhibition
	3	IC50 = 168 μM Ki = 20.7 µM
	9	90% RA at 5 mM
	10	101% RA at 0.5 mM
	11	100% RA at 0.5 mM
	12	0% RA at 5 mM 90% RA at 1 mM
	13	89% RA at 0.5 mM
	14	100% RA at 0.5 mM
	15	96% RA at 1 mM
	16	89% RA at 0.5 mM
	17	26% RA at 5 mM Nonsigmoidal curve
	18	69% RA at 5 mM 87% RA at 1.5 mM
	19	50% RA at 5 mM 90% RA at 1.5 mM
	20	100% RA at 0.5 mM
	21	96% RA at 0.5 mM
	22	100% RA at 0.5 mM
	23	94% RA at 0.5 mM
	D-Cyclo-serine	IC50 = 108 μM

Table 4. The breakdown of GlideScore scores into individual contributions, only non-zero terms are shown.

	XP GScore	XP HBond	XP PhobEn	XP LowMW	XP LipophilicEvdW	XP Electro	XP Zpotr
Glide XP (original) pose	–12.549	–0.76	–2.00	–0.5	–1.664	–5.125	–2.5
QPLD pose	–12.009	–0.57	–2.05	–0.5	–1.261	–5.125	–2.5