delivering PCPA directly to the active site of LSD1, candidate LSD1 inactivators
were designed (Fig. 5b), in which PCPA is coupled to a lysine carrier moiety at the
nitrogen atom. Because methylated lysine is the substrate of LSD1, it is expected that
the lysine moiety of the candidate inactivator would be efficiently recognized by
LSD1, which would lead to high selectivity over MAO-A and MAO-B. After the
PCPA moiety of the candidate inactivator is carried to the active site of LSD1, it is
expected that the PCPA moiety would inactivate LSD1 in a similar manner to PCPA
itself, namely, through single-electron transfer, radical opening of the cyclopropyl
ring, and covalent bond formation with FAD (Fig. 5b). Then, the lysine moiety is
expected to be released through the hydrolysis of the imine intermediate (Fig. 5b).
Thus, the lysine moiety of the candidate inactivator serves as a carrier that delivers
PCPA into the active site of LSD1 selectively and efficiently.
Initially, as a proof of concept study, PCPA-Lys-4 H3-21 (Fig. 5c), which bears a
PCPA moiety at Lys-4 of a 21-amino-acid LSD1 substrate peptide (H3-21), was
designed and prepared. As expected, PCPA-Lys-4 H3-21 strongly inhibited LSD1
with an IC 50 of 0.16 μM in a time- and concentration-dependent manner, but did not
inhibit MAO-A or MAO-B (IC 50 > 100 μM). However, PCPA-Lys-4 H3-21 showed
only weak antiproliferative activity in cancer cells where LSD1 was overexpressed.
It was speculated that PCPA-Lys-4 H3-21 has poor membrane permeability, likely
as a result of the high polarity of its peptide structure. Thus, based on this proof of
concept of the LSD1-targeted PCPA delivery strategy, this strategy was applied to
the design of nonpeptide, small-molecule LSD1 inactivators that show activity in
cell-based assays. A number of candidate small-molecule, drug-delivery-type LSD1
inactivators were designed and synthesized guided by the X-ray crystal structure of
LSD1, and NCD38 was eventually identified as a potent and selective LSD1
inactivator (Figs. 2 and 5c). NCD38 also showed potent antiproliferative activity
in solid cancer cells.
In addition, the LSD1 inactivation mechanism was investigated to confirm that
NCD38 indeed inhibits LSD1 by delivering PCPA to the LSD1 active site (Fig. 5b).
Kinetic analysis revealed that NCD38 is a time-dependent LSD1 inactivator, in
accordance with the irreversible mechanism we proposed (Fig. 5b). The kinetic
parameters of NCD38 are shown in Fig. 5d. The k inact /K i value of NCD38 is much
larger than that of PCPA, thus confirming that NCD38 is a much more potent LSD1
inactivator than PCPA. MALDI MS analysis of the inactivated mixture of LSD1
with NCD38 was also performed. Peaks with m/z 918 and 900, corresponding to the
FAD-PCPA adduct and the dehydrated adduct, respectively, were observed in the
mixture of LSD1/NCD38. The lysine moiety released from LSD1/NCD38 was also
detected. These mechanistic data strongly support the idea that NCD38 inhibits
LSD1 through the efficient and selective delivery of PCPA to the active site of
LSD1 with the assistance of its lysine moiety (Fig. 5b).
Interestingly, a recent report has shown that NCD38 derivatives inhibit LSD1 in
preference to LSD2 [61].
Recently, it has been reported that NCD38 inhibits the growth of MLL-AF9
leukemia as well as erythroleukemia, megakaryoblastic leukemia, and myelodysplastic
syndrome (MDS) overt leukemia cells in the concentration range in which normal
Lysine-Specific Histone Demethylases 1/2 (LSD1/2) and Their Inhibitors
209
were designed (Fig. 5b), in which PCPA is coupled to a lysine carrier moiety at the
nitrogen atom. Because methylated lysine is the substrate of LSD1, it is expected that
the lysine moiety of the candidate inactivator would be efficiently recognized by
LSD1, which would lead to high selectivity over MAO-A and MAO-B. After the
PCPA moiety of the candidate inactivator is carried to the active site of LSD1, it is
expected that the PCPA moiety would inactivate LSD1 in a similar manner to PCPA
itself, namely, through single-electron transfer, radical opening of the cyclopropyl
ring, and covalent bond formation with FAD (Fig. 5b). Then, the lysine moiety is
expected to be released through the hydrolysis of the imine intermediate (Fig. 5b).
Thus, the lysine moiety of the candidate inactivator serves as a carrier that delivers
PCPA into the active site of LSD1 selectively and efficiently.
Initially, as a proof of concept study, PCPA-Lys-4 H3-21 (Fig. 5c), which bears a
PCPA moiety at Lys-4 of a 21-amino-acid LSD1 substrate peptide (H3-21), was
designed and prepared. As expected, PCPA-Lys-4 H3-21 strongly inhibited LSD1
with an IC 50 of 0.16 μM in a time- and concentration-dependent manner, but did not
inhibit MAO-A or MAO-B (IC 50 > 100 μM). However, PCPA-Lys-4 H3-21 showed
only weak antiproliferative activity in cancer cells where LSD1 was overexpressed.
It was speculated that PCPA-Lys-4 H3-21 has poor membrane permeability, likely
as a result of the high polarity of its peptide structure. Thus, based on this proof of
concept of the LSD1-targeted PCPA delivery strategy, this strategy was applied to
the design of nonpeptide, small-molecule LSD1 inactivators that show activity in
cell-based assays. A number of candidate small-molecule, drug-delivery-type LSD1
inactivators were designed and synthesized guided by the X-ray crystal structure of
LSD1, and NCD38 was eventually identified as a potent and selective LSD1
inactivator (Figs. 2 and 5c). NCD38 also showed potent antiproliferative activity
in solid cancer cells.
In addition, the LSD1 inactivation mechanism was investigated to confirm that
NCD38 indeed inhibits LSD1 by delivering PCPA to the LSD1 active site (Fig. 5b).
Kinetic analysis revealed that NCD38 is a time-dependent LSD1 inactivator, in
accordance with the irreversible mechanism we proposed (Fig. 5b). The kinetic
parameters of NCD38 are shown in Fig. 5d. The k inact /K i value of NCD38 is much
larger than that of PCPA, thus confirming that NCD38 is a much more potent LSD1
inactivator than PCPA. MALDI MS analysis of the inactivated mixture of LSD1
with NCD38 was also performed. Peaks with m/z 918 and 900, corresponding to the
FAD-PCPA adduct and the dehydrated adduct, respectively, were observed in the
mixture of LSD1/NCD38. The lysine moiety released from LSD1/NCD38 was also
detected. These mechanistic data strongly support the idea that NCD38 inhibits
LSD1 through the efficient and selective delivery of PCPA to the active site of
LSD1 with the assistance of its lysine moiety (Fig. 5b).
Interestingly, a recent report has shown that NCD38 derivatives inhibit LSD1 in
preference to LSD2 [61].
Recently, it has been reported that NCD38 inhibits the growth of MLL-AF9
leukemia as well as erythroleukemia, megakaryoblastic leukemia, and myelodysplastic
syndrome (MDS) overt leukemia cells in the concentration range in which normal
Lysine-Specific Histone Demethylases 1/2 (LSD1/2) and Their Inhibitors
209