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Antitubercular Isoniazid and Drug Resistance of Mycobacterium tuberculosis

— A Review

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Arch. Pharm. Pharm. Med. Chem. 2002, 11, 511–525 Isoniazid Activation and Resistance 511

Thomas Sciora, Antitubercular Isoniazid and Drug Resistance of

Iván Meneses Moralesb,
Solón Javier Garcés Eiseleb, c, Mycobacterium tuberculosis – A Review
David Domeyerd,
Stefan Lauferd Isoniazid is one of the most potent drugs available for tuberculosis treatment. As a
pro-drug it requires activation, which is performed by catalase/peroxidase. The ac-
a
Department of Pharmacy, tive principle, whose identity has not yet been determined unambiguously, then acts
Benemérita Universidad on at least one target molecule, the enoyl-acyl carrier protein, required for the syn-
Autónoma de Puebla, thesis of the vital mycolic acids present in the cell wall of the bacterium. Some other
Puebla, Pue. 72570, México targets have been proposed in order to explain the unusual potency of isoniazid;
b
Department of Chemistry however, the supporting data are still controversial. We thoroughly discuss the ac-
and Biology, tion of isoniazid, resistance mechanisms, and the possible active product, which in-
Universidad de las cludes an isonicotinic acid-NADH adduct as well as a meta-isomer of NADH. Both
Américas-Puebla, structures have been probed positively in a 3D modeling analysis.
San Andrés Cholula,
Pue. 72820, México
c
Department of Molecular Keywords: Mycobacterium tuberculosis: Isoniazid; Isonicotinic acid; Drug resist-
Biology, ance; katG; inhA
Laboratorios Clínicos de
Puebla,
Received: August 8, 2001 [FP620]
Puebla, Pue. 72530, México
d
Pharmaceutical Institute,
University of Tübingen,
72076 Tübingen, Germany

Introduction
Tuberculosis is an infectious chronic illness of worldwide
dimensions affecting mostly patients in poor living condi-
tions. Its causal agent is mainly Mycobacterium tubercu-
losis (Mt), a slim, aerobic, acid-resistant microorganism.
Despite intensive treatment with available drugs such as
para-aminosalicylic acid (discovered 1946, now rarely
used), rifampicin, isoniazid (isonicotinic acid hydrazide,
INH), ethionamide, streptomycin, pyrazinamide, and
Epidemiology
Tuberculosis is a worldwide public health problem and is
usually transmitted by droplet infection. Approximately
one third of the earth’s population is infected with Myco-
bacterium tuberculosis bacteria. Although less than ten
per cent become sick, in 1995 more humans died of tu-
berculosis than ever before. An increasing drug resist-
ance problem together with the HIV-induced acquired
immunodeficiency syndrome is leading to further epi-

ethambutol, for almost fifty years, disease control has
not been successful due to mistreatment and insufficient
knowledge, thus involuntarily selecting resistant cul-
tures. First choice mono-medication, for example with
isoniazid, faces severe limitations because of the drug
resistance shown by Mycobacterium tuberculosis. Ge-
netic evidence exists that certain alterations in mycobac-
terial genes like katG and inhA are mainly responsible for
the resistance presented towards INH. This contribution
reviews the scientific literature relating to the elucidation
of the pharmacological mechanism of action of isoniazid
and genetic mutations which lead to drug resistance of
Mycobacterium tuberculosis.
Correspondence: Solón Javier Garcés Eisele, Department of
Chemistry and Biology, Universidad de las Américas-
Puebla, San Andrés Cholula, Pue. 72820, México. Phone:
+52 222 229 2414, fax: +52 222 229 2419, e-mail:
jgarces@mail.udlap.mx
Review
demic spread among the population.The common infec-
tion agent is M. tuberculosis, more rarely M. bovis or M.
africanum.They are also acid-resistant and morphologi-
cally similar to M. tuberculosis. However, they differ sig-
nificantly in their proliferation kinetics. In about 85 per
cent of all clinical cases the lung is concerned. With the
increase of the HIV infections, however, the proportion of
extra-pulmonary types rises. In 1997, eight million new
cases were assessed, adding to 16 million cases al-
ready registered. Of the latter, approximately two million
could have died in the meantime [1]. Some 80 % of all pa-
tients were registered in 22 countries. Five nations be-
longing to the southeast region of Africa make up more
than half of them. Not surprisingly, nine out of ten of the
highest incidences are located in this continent [2]. This
situation is likely to deteriorate in the future, with annual
disease rates expected to rise from 8.0 million in 1997 to
10.2 million per year by 2005 [3]. This alarming sign of

© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0365-6233/11/0511 $ 17.50+.50/0
512 Scior et al.
spreading of endemic disease and the fact that drug re-
sistant cultures have emerged on a massive scale un-
derscore the importance of studying molecular mecha-
nisms of drug action and those involved in the pharma-
cological resistance of Mycobacterium tuberculosis [4].
In clinical terms tuberculosis is divided into primary (I)
and reactivated forms (II).
(I) The typical source of a primary infection is a person
with pulmonary tuberculosis producing up to 3,000 aero-
sol droplets per cough attack. Today, a primary infection
need not be fatal and can be treated with appropriate
chemotherapy. However, on occasions (AIDS etc.) the
illness will not stay localized but progresses and the per-
son will suffer from secondary complications. Generally,
incorporated mycobacteria can survive after phagocyto-
sis by macrophages.To do so, they must be able to resist
many cellular defense strategies in the human body: re-
active oxygen species and nitrogen compounds as well
as enzyme activities such as, peroxidase, phosphatase,
hydrolase, protease, lysozyme, and lipase. In particular,
the lipid-rich cell wall plays an important role as protec-
tive layer against aggressive lysosomal factors from bac-
teria and diffusion barrier for drugs.
(II) Reactivated tuberculosis is a recurrence of the pul-
monary form after sensitization to M. tuberculosis during
the primary infection: after an interval of latency, a cellu-
lar immune response leads to the formation of tissue tu-
mors (granuloma).These highly characteristic pathologi-
cal lesions are also known as tubercles. Tissue destruc-
tion (fibrosis) presents focal areas with dead cells
(necrosis), forming a friable and amorphous granular
mass, cheese-like in appearance. Often these focuses
show spontaneous remission; otherwise they become
calcified. However, on occasion such material is liberat-
ed into a bronchus of the respiratory tract and is expecto-
rated, after which, the aspiration can cause inflammation
of the lungs, known as tuberculous pneumonia.If not, the
material persists in cavities as encapsulated tuberculo-
sis [5].
Drug resistant tuberculosis
All forms of drug resistance are entirely iatrogenic in na-
ture, i.e. acquired during pharmacotherapy. The most
common errors leading to the selection of drug resistant
strains among microorganisms are manifold: (i) adminis-
tration of a single drug (instead of a combination), (ii)
sub-therapeutic dosage below the pharmacologically re-
quired minimum inhibiting concentration (MIC), (iii) treat-
ment is applied for too short a period of time, (iv) non-
compliance of patients, (v) socio-economical problems
under which poor people strive to obtain medical assist-
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525
ance, (vi) insufficient regional drug availability, (vii) lack
of dispensation facilities and professional pharmaceuti-
cal care [6].
A typical clinical case of drug resistant tuberculosis
would be one of the pulmonary type, in which the patient
excretes resistant bacilli. Resistance means lack of any
anti-infectious effects for first choice medication at least
(in decreasing/increasing order of efficiency/toxicity, re-
spectively): isoniazid (isonicotinic acid hydrazide, INH),
rifampicin (rifampin, RMP), pyrazinamide (PZA), etham-
butol (EMB), and streptomycin (SM). Second-line drugs
are protionamide (PTA), ethionamide (ETA), thiaceta-
zone, thiocarlide, cycloserine, capreomycine, and para-
aminosalicylic acid (PAA), which are all bacteriostatic in
action whereas primary-line drugs are bactericidal, with
the exception of EMB at low concentrations. Some form
part of the recommended treatment for tuberculosis is-
sued by the World Health Organization (WHO) [7]. Ra-
tional use of these drugs depends on the sensitivity of
the germs. After determining an antibiotic profile (micro-
biological diagnostics), established combinations are
applied. The pharmacotherapeutical goal is to wipe out
the germs (diagnosed as sputum conversion: reduction
of colonies) or prevention of recurrence (relapse). Due to
the required long-duration (several months to years) ad-
ministration of antituberculous agents, resistance has
rapidly developed in 5–10 % of the strains (1–3 % multi-
drug resistant disease). To avoid selection of even more
primarily resistant bacteria the WHO recommends a
treatment scheme within their DOTS program com-
posed of a two-month treatment with a combination of 4
antibiotics including isoniazid, rifampicin, and pyrazin-
amide, complemented with either streptomycin or
ethambutol, followed by a 6-month treatment with isoni-
azid combined with either ethionamide or ethambutol, or
a 4-month treatment with isoniazid and rifampicin [8, 9].
Persistence or relapse after the primary (triple or quad-
ruple) therapy will be treated in turn by another threefold
or fourfold long-duration therapy (more than a year)
changing at least two drugs taken during the primary in-
fection. Each total daily dose is calculated on the basis of
body weight and can be administered orally once a day
except for streptomycin (parenteral).This scheme forms
part of the “DOTS-plus” strategy developed by the WHO
[10].
Isoniazid resistance
Mycobacteria have small water-filled pores, which are
used by smaller hydrophilic molecules such as isoniazid
(log Pexp = –0.70; [11], p-aminosalicylic acid (log Pexp =
+0.89; [12]), or protionamide (log Pest = +0.62; [13]) for
passive penetration; in contrast, larger, sufficiently hy-
drophobic drugs such as rifampicin can only diffuse
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525
along the mycolic acid layers.The unusually hydrophobic
cell wall of certain mycobacteria, such as M. leprae, rep-
resents a crucial diffusion barrier for polar and/or mainly
dissociated chemotherapeutic agents to such extent that
they may even become ineffective despite their pharma-
codynamic potency [13].
Shortly after the introduction of isoniazid chemotherapy
in 1952 [14], mycobacteria resistant to this antitubercu-
lous agent were described [15]. Nevertheless, INH has
become the basic medication [16]. It is therefore impera-
tive to know more about this drug’s molecular mecha-
nism of action, in order to prevent further spread of isoni-
azid resistance among M. tuberculosis.Winder suggest-
ed in 1964 that point mutations could lead to INH resist-
ance [17].Two years later Bekierkunst related INH activi-
ty to decrease of NAD(H) concentrations in Mt bacteria
[18]. INH has a bactericidal effect on proliferating germs,
i.e. by interfering in mycolic acid biosynthesis [19]. Cata-
lase-peroxidase negative germs are resistant [20]. Sub-
sequently, INH may be considered a prodrug assuming
that it is only activated by peroxidase [21]. Indeed, a pos-
sible resistance mechanism had been proposed [22].It is
based on the molecular interaction between INH and the
InhA enzyme from the fatty acid synthesis cascade (2-
trans-Enoyl-ACP-Reductase), which is coded by the
gene inhA. Another observation in highly resistant Mt
germs was made earlier: catalase peroxidase (KatG)
coding gene (katG) is either mutated or deleted [20,
23–25]. Just recently, this phenomenon was also as-
cribed to a third protein, denominated KasA (coded
Figure 1.Reaction of anionic or radical isonicotinic acyl agents with NADH locking reductase activity of InhA [31].Viable
(S) enantiomer depicted (see Figure 4). The asymmetric carbon atom is marked by an asterisk.
Isoniazid Activation and Resistance 513
by the gene kasA), because alterations in the expression
of this protein seem to render isoniazid administration in-
effective [26, 27].
KatG gene
KatG of M. tuberculosis (MtKatG) comprises a hemopro-
tein and is classified as oxidoreductase (peroxidase). It
is conserved in several microorganisms and many of
them are not especially sensitive to isoniazid. Purified
MtKatG is a dimeric enzyme of 80 kDa subunits, which
contains one heme group per subunit [28], connected by
a stabilizing disulfide bridge involving the Cys-20 of each
subunit [29].
INH is recognized as a prodrug [21, 30].Through the par-
ticipation of MtKatG, this precursor is converted into
highly reactive principles capable of acylation or oxida-
tion of different amino acids in proteins of microorgan-
isms [21]. Thus, we understand the role of MtKatG as a
bacterial enzymatic oxidizer, which activates the pro-
drug isoniazid under aerobic conditions.The main meta-
bolic products are identified as isonicotinic acid, isonico-
tinamide, and pyridine-4-carboxyaldehyde [21]. Other
sources suggest (see Figure 1) that oxidation and acyla-
tion are due to biotransformation of an acyl-isonicotinic
anion or an isonicotinic acyl radical, respectively [31].
Earlier, Walt and coworkers [32] excluded experimental-
ly under non-enzymatic conditions an iron-catalyzed hy-
droxyl radical mediated oxidation of isoniazid from the
discussion of the mechanism.
514 Scior et al.
Figure 2. MtKatG catalyzed activation of isoniazid to form hydrazil radical of isoniazid, and finally isonicotinic acyl radi-
cal [28, 31, 43, 91].
In the typical peroxidase cycle (see Figure 2), the en-
zyme in the ferric state (resting enzyme) reacts with a hy-
drogen peroxide equivalent to form Compound I (Cmpd
I), an oxyferryl iron-protoporphyrin IX:π-cation radical.
The reduction of Cmpd I by one electron produces a sec-
ond intermediate, viz. Compound II (Cmpd II), which
contains the heme-oxyferryl group but no longer the cat-
ionic radical. Finally, Cmpd II may be reduced further to
the ferric state accepting one additional electron. Evi-
dence exists for the formation of the Cmpd I of MtKatG.
Furthermore, it has been shown (see Figure 2) that this
compound reacts rapidly with INH [28, 33].The instability
of Cmpd I could result from electron transfer reactions
originating in exogenous and endogenous reducing
agents, which cause the Cmpd I to be discharged, rees-
tablishing the ferric enzyme state.The fact that the ferric
form is regenerated without the appearance of other in-
termediates suggests that these intermediates, includ-
ing the Cmpd II, are unstable [28]. Although it might have
been expected from considerations of homology to the
yeast cytochrome c peroxidase, the residue W321 does
not seem to be the site of stabilization of radicals. At
least, the mutation W321F in KatG does not alter the in-
termediates formed in the catalytic cycle of the enzyme.
However, in correlation with the observed isoniazid re-
sistance in clinically isolated katG mutants, W321 ap-
pears to be important for substrate binding. It has, fur-
thermore, been proposed to contribute to subunit inter-
actions within the dimer [34]. However, as can be seen in
Figure 8, W321 is located at the outer space, shielded by
the heme group from the dimerisation interface [35].
The presence of MtKatG protein (catalytic activity) can
be used as direct indicator of the correct functionality of
the katG gene [28, 33]. In most of the cases where the
enzyme is absent or inactive, alterations in the gene
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525
katG and resistance to INH are observed [20, 24, 25, 36].
Intriguingly, changing the amino acid arginine to leucine
in position 463 (R463L) does not affect the bioactivity of
the enzyme MtKatG but it produces resistance to INH
[24, 25]. It is reported that approximately 44 % of M.
tuberculosis culture, for which MIC in isoniazid is
ⱖ1.0 µg/mL, show the same mutation in position
(R463L) of katG gene [23]. However, the relationship be-
tween the mutation R463L and INH resistance has been
challenged by the observation that the mutation is found
in a high proportion of strains susceptible to isoniazid. In
addition, experiments with site-directed mutagenesis
have failed to show any effect of R463L substitution on
MtKatG activity, which suggests that it is rather a com-
mon polymorphism, independent of INH resistance. Fur-
thermore, it seems to be linked to the geographical origin
of the strains [29, 37–41].
Resistance frequently arises from a mutation changing
the serine at position 315 to a threonine (S315T). The
mutation is thought to introduce subtle changes in the
binding site for isoniazid as the enzyme has a lowered ef-
ficiency in converting isoniazid in comparison to the wild
type, however, both enzymes have similar catalase-per-
oxidase activities [42]. The resistance conferred by the
S315T mutation is not mediated by changes in the ability
to convert the iron atom from the ferric to the ferrous form
as the redox potential of the S315T mutant is essentially
the same as in the wild type form [43]. The distance be-
tween the iron atom in the active site of MtKatG and the
amide nitrogen of isoniazid does not change significantly
between the mutant and the wild type form, 3.8 ± 0.8 and
4.4 ± 0.9 Å, respectively.Therefore, there have to be oth-
er factors responsible for the reduced capacity of the mu-
tant in oxidizing isoniazid [44].The S315T mutant exhib-
its a reduced isoniazid oxidation rate dependent on su-
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525 Isoniazid Activation and Resistance 515

Table 1. Mutations and polymorphisms in the katG gene

associated with the INH resistance phenotype.

Codons Variants References

1 Met→Ala [93]
17 Ser→Asn [23]
63 Asp→Glu [25]
94 Asp→Ala [37]
108 His→Glu/Gln [25, 94]
120 Deletion [93]
122–125 Deletion [37, 40]
125 Insertion [93]
126 Met→Ile [23]
138 Asn→Ser [37, 94]
140 Ser→Asn [37]
142 Asp→Ala [37]
148 Leu→Ala [94]
Figure 3. Superoxide-dependent pathway for iron acti- 160 Ser→Leu [37]
vation in MtKatG and isoniazid (INH) oxidation-activation 169 Gly→Ala [23]
[43, 45]. 172 Ala→Thr [93]
180 Thr→Cys [93]
262 Thr→Arg [25]
peroxides and it has been suggested that either trace el- 264 Ala→Thr [95]
ements participate in the production of superoxides or 275 Thr→Pro [24, 29, 93]
that they are produced by an alternative source in the ab- 315 Ser→Thr/Asn [23, 29, 38, 40, 87, 88,
sence of the trace elements. In contrast, the oxidation 93, 96–104]
rate using a peroxidative pathway does not correlate with 328 Trp→Gly [37]
the resistance to isoniazid conferred by this mutation. 335 Ile→Thr [25]
This observation taken together with other results sug- 337 Tyr→Cys [95]
gest that the oxidation of isoniazid by MtKatG occurs by 350 Ala→Ser [25]
a superoxide dependent pathway (see Figure 3), and al- 409 Arg→Ala [24]
so suggest INH oxidation does not occur via peroxidative 463 Arg→Leu [23–25, 29, 37, 40]
pathways in vivo [45]. 463 Arg→His [40]
477 Codon stop [93]
The ferrous forms of the wild type and the S315T mutant
513 Insertion [93]
of MtKatG contain mixtures of hexacoordinated low spin
515 Arg→Cys [93]
and pentacoordinated high spin heme groups, whereas
550 Ala→Asp [23]
in the ferric state most of the groups are pentacoordinat-
567 Phe→Ser [93]
ed high spin heme groups, although it is possible to de-
587 Leu→Met/Pro [29, 93]
tect the hexacoordinated groups [28, 42, 46]. At room
593 Gly→Asp [37]
temperature, more hexacoordinated low spin groups can
609 Met→Ile [23]
be detected in the mutant in both forms, ferric or ferrous.
629 Gly→Ser [25]
This increase in hexacoordinated heme groups may be
695 Asp→Ala [24]
critical by reducing the access of oxygen or superoxide
710 Val→Ala [37]
to the iron atom, diminishing thereby the number of en-
714 Ala→Pro [94]
zymes capable of activating isoniazid, and in this way
717 Ala→Pro [25]
producing resistance [46]. The apparently conservative
substitution of serine by threonine is of maximum benefit
to M. tuberculosis as it reduces the activation of isoniazid
while maintaining a substantial catalase-peroxidase ac-
InhA Gene
tivity [42]. Various other sequence variants of the katG
gene have been described, some of which have been Efforts to identify targets of the activated INH species led
demonstrated to participate in the INH resistance (see to the discovery of the inhA locus, which is composed of
Table 1). two open reading frames (ORF), designated mabA (ini-
516 Scior et al.
tially orf1) and inhA, separated by a 21-bp non-coding re-
gion, one coding for a 29 kDa and the other for a 32 kDa
protein [22]. The first ORF exhibits sequence similarity
with other proteins involved in fatty acid synthesis; how-
ever, it was not associated with the INH resistance phe-
notype [47]. Subcloning studies demonstrated that the
second ORF of M.smegmatis was sufficient to confer the
isoniazid resistance phenotype [22].
The inhA gene codes for an enoyl-Acyl Carrier Protein
(ACP) denominated InhA reductase. In general ACPs
form part of fatty acid synthases as small peptidic com-
ponents that are modified when covalently bound to a
4⬘-phosphopantetheine molecule whose function is to
transport the growing chains of fatty acids among other
components of the fatty acid synthase enzyme in Myco-
bacterium tuberculosis. Long-chain enoyl-acyl carrier
protein reductase (InhA) catalyzes the reduction of 2-
trans-enoyl-ACP, a reaction dependent on redox coen-
zyme β-nicotinamide adenine dinucleotide (NADH).The
reduction is vital for the elongation of fatty acids during
the biosynthesis of bacterial cell wall, which constitutes a
thick mechanical defense shield of M. tuberculosis [22,
30].
The InhA structure has the appearance of a chair com-
posed of 7 β-sheets (B1–B7) and 8 α-helices (A1–A8).
The cofactor-binding site is a shallow cavity between the
“seat” and the “back” of the enzyme. NADH acquires an
extended conformation in the cavity with the adenine
ring oriented in parallel to the seat and the nicotinamide
portion oriented towards the back, pointing to a cavity
formed by the β-sheets B4 to B6 and the α-helices A5 to
A7 [48].
The enzymatic system containing most probably the en-
zyme InhA is the FAS-II system. It shares some proper-
ties with the InhA protein, such as its specificity for sub-
strates with at least 12 carbons, its preference for mole-
cules with 16 carbons, and its dependence on ACPs
[49].
Other genes
Mutations occurring in the katG and inhA genes only ac-
count for up to 80 % of all INH resistant organisms.
Therefore, new mechanisms of INH resistance are stud-
ied [50]. When purifying proteins of cultures treated with
isoniazid, a complex is found formed of isoniazid, AcpM,
and a β-ketoacyl-ACP synthase (KasA coded by the
gene kasA). This complex stops the elongation of fatty
acids, resulting in the precursor’s accumulation as can
be observed during treatment with isoniazid [26]. These
observations, next to in vitro observation concerning
cross-resistance, and the use of a specific inhibitor of
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525
InhA (thiolactomycine) with which the accumulation of
unsaturated y acids is provoked, suggest that the first
cellular target for isoniazid in M. tuberculosis is KasA
[27]. But there is no evidence that mutations in the gene
kasA produce high levels of resistance [51]. Subse-
quently, the mechanism proposed to explain it suggests
mutations in the promoters since the artificial over-ex-
pression of the gene kasA quintuples the resistance to
INH, but over-expression of InhA only duplicates it. Over-
expressing both genes simultaneously leads to tenfold
higher levels of resistance (synergism). Finally, an incre-
ment in the expression of InhA exercises an effect on the
operon, which contains the gene kasA, causing its over-
expression and elevating the resistance level in direct
proportion [27].
However, these results are in contrast with recent find-
ings and may need to be revised closely. Several muta-
tions of the kasA gene were found in INH resistant as
well as in sensitive strains. Moreover, in various INH re-
sistant strains with kasA mutations, additional mutations
were found in katG or inhA [26, 52–55]. Finally, the
groups of Sacchetini and Jacobs [56] report that they
were unable to reproduce the results of the over-expres-
sion studies mentioned in the previous paragraph. In-
stead these authors found a consistent 20-fold or greater
increase in INH resistance in mycobacteria containing
inhA plasmids, but were unable to find any increase in
resistance to INH with kasA plasmids; they did, however,
find resistance to thiolactomycine, a specific inhibitor of
KasA.They concluded, therefore, that InhA is the prima-
ry target of isoniazid.
Additional genes discussed in the context of INH resist-
ance are the oxyR-ahpC regulon [50, 57–59] and the
NADH dehydrogenase [60, 61].
The active form of isoniazid, including
stereo-chemical aspects
There is evidence that isoniazid oxidation in the pres-
ence of oxygen, Mn2+ ions, and InhA inactivates the en-
zyme [62–64], and that when enzyme MtKatG is added
the inactivation velocity increases 2.7 times [63]; more-
over, this isoniazid activation is MtKatG dependent [45].
Recently it has also been shown that activation may oc-
cur in the absence of Mn2+ ions and InhA as the omission
of Mn2+ results only in a 30 % reduction of the inhibitor
complex formed, which indicates that manganese in-
creases the efficiency but is not required for the activa-
tion of isoniazid by the enzyme KatG of M. tuberculosis
[65]. Isoniazid affects InhA through modification of
NADH.The precise nature of the active species of isoni-
azid is still under discussion. However, it is believed that
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525 Isoniazid Activation and Resistance 517

toxicity may be mediated in vivo by reactive radicals, as it
is clear that isonicotinic acid (at least under certain con-
ditions, see below), isonicotinamide, and pyridine-4-car-
boxaldehyde, the stable products of the enzymatic reac-
tion of MtKatG, are not toxic for M. tuberculosis at physio-
logical concentrations.The carbonyl carbon atom on the
acyl group of isonicotinic acid binds covalently to the ring
carbon in position 4 of NADH’s nicotinamide (INH-
NAD(H) adduct).The drug’s acyl group replaces the 4S-
hydrogen atom of NADH.This is the position in charge of
the transfer of hydride anions (H–) during substrate re-
duction. When the redox coenzyme NADH remains
blocked, the InhA enzyme is unable to exercise its reduc-
tive activity on substrates. It is suggested that the forma-
tion of the INH-NAD(H) adduct takes place by addition of
an acyl-isonicotinic anion to the NAD+ cation, or of a radi-
cal acyl-isonicotinic radical to a NAD · radical (see Fig-
ure 1) [31, 65-67].
The in vitro formation of the INH-NAD(H) adduct may oc-
cur spontaneously by a Minisci reaction between the
acyl radical and electron deficient heterocycles like
NAD+.The observed low binding affinity constant of InhA
for NAD+ (Ki = 4 mM) suggests the formation of the ad-
duct in solution rather than within the active site of InhA
[67]. Wilming and Johnsson observed furthermore a dy-
namic equilibrium between two forms of the adduct,
which they could not interpret. Interestingly, in a recent
study, it has been shown that the in vitro combination of
the isonicotinoyl radical and the nicotinamide part of
NAD+ results in the formation of two open and four hemi-
amidal-cyclised dihydropyridine structures [68]. Still, Ro-
zwarski and colleagues [31] presume that the isonicotin-

Figure 4. Ball-and-stick representation of three interacting amino acids on InhA together with its prosthetic group,
NADH. The Sybyl generated model (Sybyl 6.7, www.tripos.com) is taken from Brookhaven databank entry with PDB
code 1ZID [31] and color-coded by atom types: C, white; O, red; N, blue. Hydrogen atoms are omitted. According to the
situation reported in Figure 1, we generated the (S) enantiomer (golden) of the NADH-isonicotinic acid adduct. It is in-
verted on the corresponding asymmetric carbon atom (green asterisk) to its (R) form (green).Whereas severe van der
Waals repulsion around Gly192, Pro193, and Ile194 represented by their backbone (light blue tube, top of mid section)
excludes the viability of the (R) form (green 6 membered ring, above) as a possible INH-NAD(H) adduct bound to InhA,
its (S) form lies in a cavity (black zone, center) and its position is favored by pi-pi interaction with Phe149 [92].
518 Scior et al.
ic acyl group of the incoming adduct stereospecifically
replaces the (4S)-hydrogen atom in the NADH moiety
(see asterisks (*) in Figure 1 and 4). Therefore, we won-
der whether the (R)- or (S)-form of the above described
product could be hosted in the binding cavity of InhA. In
our own modeling study we show that the (R)-enantio-
meric product is stereochemically forbidden as visual-
ized in Figure 4. To our best knowledge (literature re-
search conducted in Summer 2002), there is no conclu-
sive report on the active principle from the INH activation
within the bacterial environment.
An alternative hypothesis had been put forward by
Krüger-Thiemer [69–71] and is depicted in Figure 5. It
suggests that INH enters the mycobacterium by passive
diffusion, facilitated by the hydrazide. Once inside the
cell, INH is oxidized enzymatically to INA, which at the in-
tracellular pH and given its pKa of 4.84, is nearly com-
pletely ionized and therefore cannot leave the cell. It ac-
cumulates until it displaces competitively the natural me-
tabolite nicotinic acid (NA) during the bacterial biosyn-
thesis of NAD(H).Thus, an analog of NAD(H) (meta-iso-
mer of NAD(H)) is produced [72]. Indeed, this analog is
able to bind to InhA (see Figure 6) and interacts with cat-
alytically important residues as discussed below [35].
This hypothesis is supported by various experimental
observations:
First of all, there is evidence that INA is able to substitute
NA during the synthesis of NAD. In experiments with hu-
man NADases, Zatman [73–75] observed the produc-
Figure 5. Isonicotinic acid hypothesis [21, 22, 69, 70, 72, 76].
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525
tion of a yellow pigment in a reaction mixture that con-
tained NAD, isoniazid, and enzyme at pH 9.5. The ap-
pearance of the pigment was paralleled by the decrease
of NAD, demonstrating a direct relationship between the
two processes. In addition it was observed that INH pre-
vented the accumulation of adenosine as a degradation
product of NAD, catalyzed normally by pig brain NAD-
ase, whereas nicotinamide hydrazide (NH) failed to do
so. The expected decrease in the NAD content can be
observed in M. tuberculosis bacilli when they are incu-
bated in the presence of isoniazid. This significant
change could be observed about 4 hours after the incu-
bation, and in some cases the decrease was even over
50 % [18].
Seydel’s work [76] adds evidence that the reactivity of
the pyridine nitrogen is essential for the biological activity
of the ortho substituted isonicotinic acid hydrazides. In
addition, his group could show in theoretical QSAR mod-
els that among the derivatives of INH the latter remains
the most active principle [72]. Furthermore, if adminis-
tered at pH of 5.9, INA (pKa 4.9 [77] is able to inhibit the
growth of mycobacteria. Under these conditions of acidi-
ty almost 10 % of INA is neutralized, crossing the hydro-
phobic cell wall and membranes. Under these conditions
it exhibits a MIC of 128 µM. Interestingly, resistant strains
are affected to the same extent as susceptible ones,
which confirms the observation that INH resistance is
mainly caused by deficiency of the catalase-peroxidase
system which frees INA from INH [72].
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525 Isoniazid Activation and Resistance 519

Figure 6. Ball-and-stick representation of two important amino acids for enzymatic activity on InhA together with its
prosthetic group, NADH.The Sybyl generated model is taken from the PDB file 1ZID [31] and color-coded as in Figure 4
by atom types. Hydrogen atoms are partly omitted for better viewing. Simulating the proposed meta-isomer of NAD(H)
[69, 72], where isonicotinic acid replaces nicotinic acid, we find a hydrogen bond (yellow) between its amide group and
Tyr158 under MMFF94 force field standard conditions. Since Tyr158 is experimentally shown [82] to play an important
role in natural substrate reduction, its temporary binding to a false substrate, namely the meta-isomer of NAD(H) (iso),
could explain the partial loss of enzyme activity [35], just like mutants show when the para-hydroxyl group of Tyr158 is
lost by introducing amino acids like phenylalanine and alanine [82].

The working hypothesis for development of an inhA-
based resistance model is that some mutations modify
the active site of the enzyme, diminishing the affinity of
InhA for NADH, or for the inhibiting complex. Substitution
of alanine for serine at position 94 (S94A) in InhA seems
to decrease the affinity of the mutant protein for NADH
due to a perturbation in the hydrogen-bonding network
that stabilizes NADH binding [48]. The S94A mutation
occurs in the cofactor-binding region. The hydroxyl of
Ser94 interacts via a water molecule with one of the oxy-
gen atoms of the pyrophosphate moiety of NADH in hy-
drogen bonding distance. This interaction is lost in the
mutant and the resulting reorientation of Gly14 in loop 1
causes small but potentially significant structural chang-
es [30]. Other mutations have been reported localized in
the NADH binding site of InhA that confer resistance to
isoniazid: I16T, I12V, I47T, I95P, V78A, and S94A [78,
79]. Mutations at the ribosomal binding site (RBS) of
mabA are associated with an overexpression of InhA
due to a transcriptional up-regulation of the whole inhA
operon [47].This genetic scenario could explain specific
drug resistance to isoniazid [31].This type of mutation is
more frequent in cultures of M. tuberculosis with active
KatG [24, 80]. Other residues putatively involved in the
catalytic process have been identified by sequence com-
parisons with members of the alcohol dehydrogenase
family and structural analysis. The tyrosine 158 (Y158)
plays a role in binding the substrate by interacting with its
carbonyl group [81]. Consequently, replacement ofY158
with phenylalanine (Y158F) or with alanine (Y158A) re-
520 Scior et al. Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525

sults in 24- and 1500-fold decreases in the catalytical
constant, respectively, while leaving KM for the substrate,
trans-2-dodecenoyl-CoA, unaffected. However, replace-
ment of Y158 with serine (Y158S) results in an enzyme
with wild-type activity. These data may be interpreted
such thatY158 functions as an electrophilic catalyst, sta-
bilizing the transition state for hydride transfer by hydro-
gen bonding to the substrate carbonyl [82]. Interestingly,
in our modeling study (see Figure 6), this moiety is able
to interact with the meta-isomer of NAD(H) [35].

Table 2. Available 3D structures of the InhA protein in the Brookhaven databank.

PDB Resol. Molecule Substrates Reference

1ENY 2.2 Å Enoyl-[Acyl-Carrier-Protein] Reductase NAD+ [48]

1ENZ 2.7 Å Enoyl-[Acyl-Carrier-Protein] Reductase NAD+ [48]
1ZID 2.7 Å Enoyl-[Acyl-Carrier-Protein] Reductase (1.3.1.9) Isonicotinic-acyl-NAD+ [31]
1BVR 2.8 Å Enoyl-[Acyl-Carrier-Protein] Reductase NAD+ and trans-2-Hexa- [81]
decenoyl-(N-acetyl-
cysteamine)-thioester

Figure 7. 3D-representation of InhA protein, taken from Brookhaven databank (entry code 1ZID) [31]. Amino acids are
omitted and backbone schematically depicted (helices violet, loop regions light blue, beta strands yellow arrows). The
prosthetic group, NADH, is modeled (C, white; H, light blue; O, red; N, blue). The covalently bound acyl form of INH is
represented as space-filling model.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525 Isoniazid Activation and Resistance 521

Figure 8. 3D-stereo-representation of KatG enzyme as a computed Swiss-model [85] based on 1ITK [84]. Amino acids
are omitted and the backbone is schematically depicted (green blue and mauve lines of A and B chain of KatG dimer,
respectively). We show the position of W321 (yellow, space filling). The prosthetic heme is colored according to atom
types (C, white; O, red; N, blue; Fe, green).

Figure 9. Stick representation of the active site on KatG enzyme as a computed Swiss-model [85] based on 1ITK [84].
The prosthetic heme (C, orange; Fe, red; H, light blue), as well as interacting aspartate (D94), arginine (R104), histi-
dines (H270, H 276), tryptophan (W107, W321), proline (P100) and serine (S315) are identified (C, white on amino
acids; O, red; N, blue).
522 Scior et al.
Protein structures
In 1997, X-ray crystallography (Brookhaven Databank
entry 1BVR, a complete survey of available 3D-struc-
tures dealing with InhA is given in Table 2) and mass
spectrometry revealed that the bacterial target of pro-
drug isoniazid is InhA through covalent binding of its
KatG activated form (isonicotinic acyl moiety) to the nico-
tinamide ring of nicotinamide adenine dinucleotide
(NADH) which in turn is non-covalently docked at the ac-
tive site of InhA (see Figure 4 and 7) [31]. As bactericidal
mechanism of action follows a fatal disturbance in cell
wall biosynthesis because the enzyme’s role is catalyz-
ing biosynthesis of mycolic acids in M. tuberculosis
[83].
Concerning the MtKatG enzyme, there is still no com-
plete three-dimensional structure available in the
Brookhaven Databank. However, the crystal structure of
the highly homologous (56 %) catalase-peroxidase from
Haloarcula marismortui has recently been determined
[84]. We computed a Swiss-model [85] (see Figures 8
and 9). According to its role of INH activation, the katG
gene is a major target for mutations in INH-resistant
strains [80]. Thus far, a large number of mutations have
been described, of which especially the Ser to Thr muta-
tion at position 315 (S315T) is found with a high frequen-
cy and has been shown to be associated with high levels
(MICs of 5–10 µg/mL) of INH resistance [86–88].Yama-
da and coworkers inferred a deep understanding of the
effects of the S315T mutation [84]. The mutated amino
acid is sitting at the entrance to the active site, narrowing
most probably the diameter of the channel to the heme
group, thereby blocking the access for INH but having lit-
tle effect on the diffusion of smaller substrate molecules.
On the other hand, the Arg to Leu mutation (R463L) of
katG does not guarantee INH resistance of all katG-
R463L mutated M. tuberculosis strains, i.e. INH pro-drug
activation by KatG is not suppressed in these cases
[41].
In March 2001 Powers, Hillar, and Loewen compared X-
ray absorption models of the heme-containing active site
in catalase-peroxidase KatG between M. tuberculosis
and E. coli. They reveal the same distance between lig-
ands and heme position on the one side, and different
enzymatic reactivity against INH on the other side. They
conclude, “it was not the heme iron environment that pro-
duced the differences in reactivity. ...” [89].
A multi sequence alignment study was performed to
compare the catalase-peroxidase (KatG) family to other
better-known members of the peroxidase superfamily
with a prosthetic heme group at the active site. Parts of
the sequence were ascribed to the role of access control
to the active site and side-chain engagement for redox
function of the central Fe-ion [90].
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 511–525
Conclusions
Mycobacterium tuberculosis infects over one-third of the
world’s population and causes almost three million
deaths every year worldwide. Isoniazid (INH) is used in
combination with other primary antituberculous drugs to
treat diseased patients. As to date (Summer 2002) con-
clusive evidence is gathered around the following points:
Isoniazid is a pro-drug, which is activated by a catalase-
peroxidase enzyme (KatG). The activated drug subse-
quently interacts with one or more targets: InhA (an
NADH-dependent enoyl [acyl carrier protein] reductase)
and/or KasA (a β-keto acyl carrier protein synthase).
Mutations in katG account for the majority of INH-resist-
ant clinical isolates. Mutations at the inhA locus, which
encodes enzymes involved in mycolic acid biosynthesis,
participate in INH resistance. However, mutations occur-
ring in these two genes only account for up to 80 % of all
INH resistant organisms. In the other 20 % of isolates,
the molecular mechanisms responsible for INH resist-
ance are still unknown. In the effort to identify the inhibi-
tor, there are two mayor mechanisms outlined in the liter-
ature: INH is activated by KatG, forming an acyl radical,
which in turn reacts with NAD(H) forming an adduct. Al-
ternatively, the stable product of the oxidation process,
isonicotinic acid (false substrate), replaces the nicotinic
acid moiety (natural substrate) of bacterial NAD biosyn-
thesis, thus forming an inhibitor (false prosthetic group of
InhA). At this stage of knowledge it is strongly desirable
to have experimental studies focused on in vivo bacterial
assays.
Acknowledgments
Special thanks are due to William Jacobs Jr. for discuss-
ing with us unpublished results, and to Joachim Seydel
for stimulating discussions. We are also grateful to Gerd
Helms, Katharina Bauer, David Martin at University of
Tübingen, Germany for assistance.
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International Union of Pure and
Applied Chemistry (IUPAC)
An essential step
in the preclinical phase
of drug development
Contents:
The Physicochemical Background: Fundamentals of Ionic
Equilibria • Solubility and Dissolution of Weak Acids, Bases,
and Salts • Evaluation of Solid-State Properties of Salts •
Pharmaceutical Aspects of the Drug Salt Form • Biological
Effects of the Drug Salt Form • Salt-Selection Strategies • A
Procedure For Salt Selection and Optimization • Large-Scale
Aspects of Salt Formation: Processing of Intermediates and
Final Products • Patent Aspects of Drug-Salt Formation •
Regulatory Requirements for Drug Salts in the European
Union, Japan, and the United States • Selected Procedures
for the Preparation of Pharmaceutically Acceptable Salts of
Acids and Bases • Monographs on Acids and Bases
86412015_ba
For customers in Germany, Austria, and Switzerland:
Wiley-VCH, P.O. Box 10 11 61 , D-69451 Weinheim;
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P. Heinrich Stahl / Camille G. Wermuth (Eds.)
Handbook of Pharmaceutical Salts
Properties, Selection, and Use
2002. Approx 400 pages. Hardcover. Approx E 149.00* / £ 75.00 / sFr 250.00.
ISBN 3-906390-26-8. Publication date: March 2002
*The e-Price is only valid for Germany.
O– Na+
O
NH
Cl Cl
The majority of medicinal chemists in pharmaceutical industry whose primary focus is the
design and synthesis of novel compounds as future drug entities are organic chemists for whom
salt formation is often a marginal activity restricted to the short-term objective of obtaining
crystalline material, and, because a comprehensive resource that addresses the preparation,
selection, and use of pharmaceutically active salts has not been available, may forego the
opportunities for increased efficacy and improved drug delivery provided by selection of an
optimal salt. To fill this gap in the pharmaceutical bibliography, we have gathered an international
team of seventeen authors from academia and pharmaceutical industry who, in the contributions
to this volume, present the necessary theoretical foundations as well as a wealth of detailed
practical experience in the choice of pharmaceutically active salts.
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