Tyrosinase Inhibitors
669
the series, with an IC50 of about 11 g/mL. The ethyl ester
of gentisate also met our criteria for an effective enzyme
inhibitor. The IC50 of the n-propyl ester was 29 g/mL. This
are concomitantly assessed for inhibition of mammalian
tyrosinase [1] using cell-free extracts of MelAb cells. If
candidate compounds prove efficacious and non-toxic by
these criteria, tests can be performed to determine if they
are mutagenic, using a mammalian V79 cell assay, described
here. Further testing using, for example, animal models can
then be performed as warranted. The strategy described
here is the minimum criterion for the discovery of candi-
date skin-lightening agents using in vitro methods.
is half the IC for the branched i-propyl ester, 58 g/mL,
50
suggesting that the active site prefers the unbranched
n-propyl ester shape. The IC of the n-butyl ester was 128
50
g/mL, or more than four times the value of the n-propyl
ester, and suggests that the binding site might accommo-
date up to three carbon atoms beyond the ester. Methoxy-
ethyl ester had an IC of 100 g/mL. This is smaller than
We believe that candidate skin-lightening agents should
satisfy at least the following criteria before further testing.
First, candidate compounds ought to be enzyme inhibitors,
preferably with the capacity to coordinate with the coppers
in the active site of tyrosinase. This property is likely to
confer some specificity to tyrosinase, since two-copper
complexes are rare, and copper coordination can also
increase binding and thus improve inhibition. Second,
candidate compounds must exhibit low toxicity. Unfortu-
nately, some compounds with the capacity to coordinate
metals can be very toxic, and must be ruled out. Some
compounds are non-toxic to non-melanocyte cells (e.g.
keratinocytes), but become toxic when acted upon by
tyrosinase and converted into quinones within melano-
cytes. HQ, for example, is more toxic to melanocytes than
non-melanocytes [22], but is also generally toxic and
mutagenic [6, 10, 11; this work]. Arbutin is a prodrug form
of HQ [23], and is much less toxic, but does not sufficiently
inhibit cell pigmentation or enzyme activity by the criteria
presented here. Candidate compounds must actively or
passively traverse two intact membranes at moderate con-
centrations without metabolic bioinactivation in order to
act on living melanocytes. This requirement likely elimi-
nates a large number of otherwise potentially effective
enzymatic inhibitors. For instance, we speculate that kojic
acid [24], a copper chelator with enzymatic inhibitory
activity against the free enzyme, fails to enter the cells.
Some compounds, such as magnesium L-ascorbyl-2-phos-
phate [25], are non-toxic and might work at very high
concentrations, but are not effective candidates by our
criteria. Lastly, visual microscopic inspection of compound
effects on cell morphology is an essential part of the
evaluation of candidate skin-lightening agents, as an indi-
rect index of cytotoxicity. To our knowledge, only MG
(and perhaps ethyl gentisate) satisfies all of these candidacy
criteria for a topical skin-lightening agent.
50
the value for the n-butyl ester, although the two compounds
are essentially the same size. The n-pentyl ester is the
largest compound that we tested in the ester series. It was
not effective as a tyrosinase inhibitor (IC Ͼ 200 g/mL),
50
suggesting a size limit to that part of the active-site cavity.
The differences in mass among this series had little effect
on activity (i.e. when expressed in molar terms). The
largest of these compounds (n-pentyl; 224 Da) is about
one-third more massive than the smallest (methyl; 168 Da),
yet its activity was over 20-fold less.
Column 4 of Table 2 displays MelAb cell pigmentation
IC
values for these gentisate esters. Each compound
50
inhibited melanocyte pigmentation (both synthesis and
total cell number) within the criterion we set for efficacy.
Interestingly, the n-propyl, n-butyl, and n-pentyl esters,
which are less effective tyrosinase inhibitors, were the most
effective agents of the series on cultured cells, with IC
50
values of near 15 g/mL, albeit due to increased cytotox-
icity characteristics. The next most effective depigmenta-
tion agents of the series were the methyl, ethyl, and
isopropyl esters, with IC50 values of about 30 g/mL against
intact melanocytes. Interpretation of the activities of some
of the larger esters is difficult, since many factors likely
contribute to the overall “depigmentation” of melanocyte
cultures (i.e. combination of inhibition of tyrosinase ex-
pression or activity and/or other components in the mela-
nogenesis pathway, cytostasis, and cytotoxicity).
Column 5 of Table 2 presents melanocyte cytotoxicity
results for the seven alkyl esters. Most of the compounds
were cytotoxic, and only MG, with an IC of about 119
50
g/mL, and methoxy-ethyl-ester gentisate, with an IC of
50
about 187 g/mL, met our arbitrary criterion for candidacy.
It is interesting that the methoxy-ethyl-ester, which did not
inhibit the tyrosinase enzyme, and was non-cytotoxic by
our criteria, is a depigmentation agent. However, it is not
likely via action on tyrosinase enzyme, and the reason for
this behavior is not known.
Having analyzed more than 150 compounds in detail [5,
6; this work; and unpublished results], it is our general
impression that testing at only a single concentration point,
40 g/mL, is sufficient during the primary cell-based screen.
Compounds that produce a lightening effect at this con-
centration are usually worthy of further study. Agents that
render cells with normal morphology and pigment levels at
this concentration are ineffective, whereas compounds that
are cytotoxic at this concentration are excludable. Except
for MG, which permits cell viability at this concentration,
all other alkyl esters yielded cells with some evidence of
toxicity.
DISCUSSION
In this work we presented a multi-endpoint screening
strategy for the discovery of safe and effective active
ingredients for topical skin-lightening or depigmentation
products. Candidate compounds are assessed in MelAb cell
cultures [6] for their ability to inhibit pigmentation of
melanocytes and to determine their toxicity. Compounds
that inhibit melanocyte pigmentation with low cytotoxicity