Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

Figures & data

Scheme 1. The synthesis of the hydroxamate inhibitors: (a) H-Phe-NHBzl·CF₃COOH (for 2a) or H-Leu-NHBzl·CF₃COOH (for 3a), TEA, THF; (b) H₂, 10% Pd/C, MeOH.

Table 1. Inhibition of human DPP III by dipeptidyl hydroxamic acids at pH 8.0*.

Inhibitor	Ki (μM)
H-Phe-Phe-NHOH	0.11 ± 0.01
H-Phe-Leu-NHOH	1.24 ± 0.47
H-Phe-Gly-NHOH	14.51 ± 0.61
H-Tyr-Phe-NHOH	0.103 ± 0.001

*The K_i values, which are presented as mean ± standard deviation, were determined with Arg₂–2NA as substrate as described in Methods.

Table 2. Inhibition of human and bacterial DPP III by dipeptidyl hydroxamic acids at pH 7.4*.

Inhibitor	Human DPP III Ki (μM)	Bacterial DPP III Ki (μM)
H-Phe-Phe-NHOH	0.028 ± 0.013	0.049 ± 0.006
H-Phe-Leu-NHOH	0.65 ± 0.23	0.075 ± 0.018
H-Phe-Gly-NHOH	4.63 ± 0.58	0.795 ± 0.088
H-Tyr-Phe-NHOH	0.030 ± 0.001	0.062 ± 0.002

*The K_i values, which are presented as mean ± standard deviation, were determined with Arg₂–2NA as substrate as described in Methods.

Figure 1. Multiple sequence alignment (MSA) of human (UniProt entry: Q9NY33) and B. thetaiotaomicron (UniProt: Q8A6N1) protein sequences. MSA was obtained using ClustalO (www.uniprot.org./align). A section of MSA comprising the constituents of the S1 and S2 subsite, marked with 1 and 2, below alignment is presented in the figure. Five evolutionary conserved regions of the M49 family are framed. Identical amino acid constitutents of S1 and S2 subsites are shadowed grey and those which differ are printed in white on black.

Table 3. Constituents of the substrate binding subsites S2 and S1 in human and bacterial DPP III *.

S2		S1
Human	Bt	Human	Bt
Glu316	+	Tyr318	+
Ile390	+	Glu329	+
Asn391	+	Phe381	Leu372
Ile392	Leu386	Pro387	Ala381
Asn394	+	Gly389	+
Asp396	Asn390	Ile390	+
Arg399	+	His450	+
His455	+	Glu508	+
Trp495	-	His568	+
Asp496	-
Ser504	+
Glu507	+
Glu508	+

*Residues forming the subsites in human DPP III were defined by the crystal structure of a human DPP III – tynorphin complex¹⁸. Structurally equivalent residues in Bacteroides thetaiotaomicron (Bt) enzyme were found by the multiple sequence alignment. +, conserved residue; - gap in the MSA.

Bezerra GA, Dobrovetsky E, Viertlmayr R, et al. Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III. Proc Natl Acad Sci USA 2012;109:6525–30

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