Lupine Publishers | Scholarly Journal of Food and Nutrition
Abstract
Entecavir is an oral antiviral drug used in the
treatment of hepatitis B infection. Entecavir is a guanosine nucleoside
analogue with selective activity against hepatitis B virus (HBV), which
inhibits reverse transcription, Hepatitis B virus (HBV) is highly endemic in
South Africa and across sub-Saharan Africa, where around 8% of people are
chronically infected, and rates of HBV-related liver cancer are some of the
highest in the world. Globally, viral hepatitis causes approximately 1.3
million deaths every year-more than either malaria or tuberculosis-with around
240 million people chronically infected with HBV1. The currently available
anti-HBV drugs show potent antiviral activity in patients with chronic
hepatitis B; however, the resistance and cross-resistance to the drugs is a
major obstacle in long-term treatment. Many studies have been conducted to
understand the molecular basis of drug resistance, and the mechanistic
characterization and molecular modeling of anti-HBV drugs complexed with HBV RT
have been reported.
Although the three-dimensional X-ray structure
of HBV polymerase is not available, its homology model has been reported using
the X-ray structure of HIV RT as a template [1-5]. Even though the homology
models may not be accurate due to the low sequence homology between the overall
HIV and HBV polymerase, the sequence conservation between the RT domains of HIV
and HBV polymerase enables molecular modeling of HBV RT [6]. In particular, the
residues around the active site that are responsible for recognizing the
template-primer or an incoming nucleoside triphosphate are highly conserved.
Nucleoside analogue HBV polymerase inhibitors cause chain termination after
incorporation into the growing chain in the active site of HBV polymerase and
consequently inhibit viral reverse transcriptase. Thus, the HBV homology model
structure based on the crystal structure of HIV polymerase serves as a useful
guide for understanding the molecular basis of HBV resistance to drugs.
Introduction
The initial patent on entecavir expired in South
Africa in 2011 ZA 1991/07894. Current status available on: http://patentsearch.
cipc.co.za/, which should have permitted lower-cost generic competitors to
enter the market. However, South Africa granted BMS three additional patents on
entecavir that only expire between 2022 and 2026. Two of these patents have
lapsed-meaning BMS has not paid the renewal fees, and they cannot be
enforced-while one patent covering a lower dosage form of entecavir remains in
force. This patent is currently under litigation in India Basheer S. BMS
Hepatitis Patent Invalidated: A Viral Effect for India? http://spicyip.
com/2013/02/bms-hepatitis-patent-invalidated-viral.html, but because it is in
force in South Africa, generic suppliers may be discouraged from bringing their
low-dose products to market.
A more recent patent on entecavir has not yet
been received or processed by the Patents Office, but it could be filed up
until the end of 2014 Patent number: WO/2013/177672. Current status available
on pa-tentscope.wipo.int. This patent covers the manufacturing process of
entecavir, and is an example of patent evergreening where companies file
patents on minor changes to an existing drug to maintain patent protection and
block competition. The same patent was recently overturned in the United States
for failing to meet the criteria of inventive step. However, in South Africa,
since no examination of patent applications occurs, if the patent is filed, it
is likely to be granted to BMS. So long as BMS maintains a monopoly on
entecavir in South Africa, the price is likely to remain high, and entecavir
will remain out of reach for those who need it. But the crystalline forms of
entecavir and its performances are not researched and reported in the
above-mentioned patent.
The review relates to analogues of
2′-cyclopentyl deoxyguanosine, especially relates to entecavir, its preparation
and the pharmaceutical composition and uses therefore this method was capable
to detect Entecavir and its diastereomeric impurities at a level below 0.009%
with respect to test concentration of 500μgml-1 for a 20μL injection volume.
The method has shown good, consistent recoveries for diastereomeric impurities
(95- 105%). The test solution was found to be stable in the diluent for 48h. The
drug was subjected to stress conditions. The mass balance was found close to
99.5%. Entecavir also helps to prevent the hepatitis B virus from multiplying
and infecting new liver cells, is also indicated for the treatment of chronic
hepatitis B in adults with HIV/AIDS infection.
Method Details
C18 stationary phase
(150 x 4.6mm, 3.5microns particles) with the economical and simple mobile phase
combination delivered in an isocratic mode at a flow rate of 1.0mlmin-1 at 254nm. In the developed method, the resolution
between Entecavir and its diastereomeric impurities was found to be greater
than 2.0. Regression analysis shows an r2 value (correlation coefficient)
greater than 0.999 for Entecavir and for its diastereomeric impurities
Discussion
The chemical name for
entecavir is 2-amino-I,9- dihydro-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-
methylenecyclopentyl)-6H-purin-6-one, monohydrate. Its molecular formula is C12H15N5O3.H2O, which corresponds to a molecular weight of
295.3 (Figure 1). Entecavir, BMS-200475,
SQ-34676(1S,3R,4S)-9-[4-Hydroxy-3-(hydroxymethyl)-2- methylenecyclopentyl]
guanine CAS-42217-69-4, 209216-23-9 (monohydrate). Anti-Hepatitis Virus Drugs,
Anti-Infective Therapy, Antiviral Drugs -Phase III This research article
describes a simple, sensitive and cost effective mobile phase method for
determination/ quantitation of diastereomeric impurities of Entecavir in drug
substances as well as in drug products. Comparison of different techniques. The
work also includes the method development and the complete validation [7] as
per ICH guidelines. Hitherto; there is no article for the quantification and
determination of diastereomeric impurities of Entecavir in drug substances and
drug products. This is a novel and sensitive method for the diastereomeric
impurities in Entecavir using HPLC (Figure 2).
Patent information
Bristol-Myers Squibb was the original patent
holder for Baraclude, the brand name of entecavir in the US and Canada. The
drug patent expiration for Baraclude was in 2015. On August 26, 2014, Teva
Pharmaceuticals USA gained FDA approval for generic equivalents of Baraclude
0.5 mg and 1 mg tablets; Hetero Labs received such approval on August 21, 2015;
and Aurobindo Pharma on August 26, 2015. Chronic hepatitis B virus infection is
one of the most severe liver diseases in morbidity and death rate in the
worldwide range. At present, pharmaceuticals for treating chronic hepatitis B
(CHB) virus infection are classified to interferon α and nucleoside/nucleotide
analogue, i.e. Lamivudine and Adefovir. However, these pharmaceuticals cannot
meet needs for doctors and patients in treating chronic hepatitis B virus
infection because of their respective limitation.
Entecavir (ETV) is
referred to as 2′-cyclopentyl deoxyguanosine (BMS2000475) which belongs to
analogues of Guanine nucleotide and is phosphorylated to form an active triple
phosphate in vivo. The triple phosphate of entecavir inhibits HBV polymerase by
competition with 2′-deoxyguanosine-5′-triphosphate as a nature substrate of HBV
polymerase, so as to achieve the purpose of effectively treating chronic
hepatitis B virus infection and have strong anti-HBV effects. Entecavir,
[1S-(1α,3α,4β)]-2-amino-1,9-dihydro-
9-[4-hydroxy-3-hydroxymethyl]-2-methylenecyclopentyl]-6Hpurin- 6-one,
monohydrate, and has the molecular formula of C12H15N5O3.H2O and the molecular weight of 295.3. Its
structural formula is as follows:
Entecavir was successfully developed by
Bristol-Myers Squibb Co. of USA first and the trademark of the product
formulation is Baraclude™, including two types of formulations of tablet and
oral solution having 0.5mg and 1mg of dosage. Chinese publication No. CN1310999
made by COLONNO, Richard J, et al discloses a low amount of entecavir and uses
of the composition containing entecavir in combination with other
pharmaceutically active substances for treating hepatitis B virus infection,
however, the entecavir is non-crystal. In addition, its oral formulations such
as tablet and capsule are made by a boiling granulating process. The process is
too complicated to control quality of products during humidity heat treatment
even though ensuring uniform distribution of the active ingredients (Figure 3).
Synthesis of BMS-200475 (EN: 182634) The
regioselective reaction of cyclopentadiene (I) and sodium (1) or commercial
sodium cyclopentadienide (II) (2, 3) with benzyl chloromethyl ether (III) by
means of the chiral catalyst (-)-diisopinocampheylborane in THF, followed by
hydroxylation with H2O2/NaOH, gives
(1S-trans)-2-(benzyloxymethyl)-3-cyclopenten-1-ol (IV), which is
regioselectively epoxidized with tert-butyl hydroperoxide and
vanadylacetylacetonate in 2,2,4-trimethylpentane, yielding
[1S-(1alpha,2alpha,3beta,5alpha)-2-(benzyloxymethyl)-6-
oxabicyclo[3.1.0]hexan-3-ol (V). The protection of (V) with benzyl bromide and
NaH affords the corresponding ether (VI), which is condensed with
6-O-benzylguanine (VII) by means of LiH in DMF to give the guanine derivative
(VIII). The protection of the amino group of (VIII) with
4-methoxyphenyl(diphenyl)chloromethane (IX), TEA and DMAP in dichloromethane
gives intermediate (X), which is oxidized at the free hydroxyl group with methylphosphonic
acid, DCC and oxalic acid in DMSO (1) or Dess Martin periodinane in
dichloromethane (2, 3), yielding the cyclopentanone derivative (XI).
The reaction of (XI)
with (i) Zn/TiCl4/CH2Br2 complex in THF/
CH2Cl2 (1), (ii) activated Zn/PbCl2/CH2I2/TiCl4 in THF/CH2Cl2 (2), (iii) Nysted reagent/TiCl4 in THF/CH2Cl2 (2, 3) or (iv) Tebbe reagent in toluene
(2) affords the corresponding methylene derivative (XII), which is partially
deprotected with 3N HCl in hot THF, providing the dibenzylated compound (XI).
Finally, this compound is treated with BCl3 in dichloromethane (1-3). (Scheme
18263401a) Description Hydrate, m.p. >220 ᴼC, alpha (22,D) +34?(c 0.3,
water) (1); monohydrate, white crystalline solid, m.p. 234-6 ᴼC (decomp.) for
the bulk sample and m.p. 255 ᴼC (decomp.) for an analytical sample
recrystallized from water, alpha(D) +33.2?(c 0.3, water) (2); alpha(D) +35.0?(c
0.38, water) (3). Manufacturer Bristol-Myers Squibb Co. (US). References1.
Zahler R, Slusarchyk WA (Bristol- Myers Squibb Co). Hydroxymethyl
(methylenecyclopentyl) purines and pyrimidines. EP 481754, JP 92282373, US
5206244. 2. Bisacchi GS, Sundeen JE(Bristol-Myers Squibb Co).
Improved process for preparing the antiviral
agent [1S-(1alpha,3alpha,4beta)]-2-amino-1,9-dihydro-9-[4-hydroxy- 3-(hydroxymethyl)-2-methylenecyclopentyl]-6H-purin-6-one.
WO 9809964. 3. Bisacchi GS, Chao ST, Bachard C, et al. BMS- 200475, a novel
carboxylic 2’-deoxyguanosine analog with potent and selective anti-hepatitis B
virus activity in vitro. Bioorged Chem Lett 1997, 7: 127-32.EP 0481754; JP
1992282373; US 5206244, Zahler R, Slusarchyk WA (Bristol-Myers Squibb Co.),
Hydroxymethyl(methylenecyclopentyl)purines and pyrimidines. EP 0481754, JP
1992282373, US 5206244, EP 0481754, JP 1992282373, US 5206244,():WO 9809964
Preparation Method
a) The reaction steps: l is prepared with a short fractionating column (filler may
be added) the atmospheric distillation unit, the receiving flask was added
anhydrous calcium chloride, and placed in an ice-water bath polymerization (monomer
cyclopentadiene rt shall cryopreservation) and tail have to take over the
drying tower. Dicyclopentadiene was added to the three-neck flask, the system
micro nitrogen, warmed slowly with stirring to 180 °C, holding the distillation
gas inlet temperature does not exceed 42°C, to give a final monomer
cyclopentadiene (cryopreservation).
b) Preparation 2: was added to the kettle in dry xylene, sodium metal to
surface oxidation, micro nitrogen, warmed to and stirred at 120~ 150 °C, the
sodium was dissolved, with vigorous stirring, sodium dispersed into sodium
sand, stirring was stopped, the system was returned to room temperature, sodium
sand cured, removing surface xylene, replacement with a suitable amount of
anhydrous THF three times, finally dry THF was added protection, backup. In the
micro nitrogen, tetrahydrofuran ice-water bath - Sodium sand cooling 0~10 °C,
the prepared cyclopentadiene monomer was slowly added dropwise to a
tetrahydrofuran - sodium sand system, control the temperature not exceeding 10
°C, After the dropwise addition, the ice-water bath was removed, allowed to
warm to rt naturally and stirred for about 3 hours, and sediment sodium is
consumed, to give a final solution of sodium cyclopentadiene reddish.
c) Preparation 3: The dimethylphenyl chlorosilane and anhydrous
tetrahydrofuran were added to the reaction kettle, micro under N2, and the
system was cooled to -70 °C or less, dropwise addition of 2, was added dropwise
to control the temperature - below 70 °C, addition was complete the mixture was
stirred at -70 °C or less incubated for about 3 hours, TLC the reaction was
complete, was naturally warmed to 0 °C, was slowly added to ice water, stirred,
allowed to stand, and the organic phase , washed with saturated sodium
bicarbonate solution and extracted with n-hexane with, dried over anhydrous
sodium sulfate, and concentrated under reduced pressure at 65 °C, the final 3
to give a dark yellow oil.
d) The preparation of 4: 3 and the reactor was added n-hexane cooled to -10 °C,
quickly dichloroacetyl chloride dropwise wherein continued stirring,
triethylamine and a mixture of n-hexane was slowly added to the system
dropwise, the temperature was kept at 5 °C or less, addition was complete, the
reaction at 0~4°C for about 4 hours, then warmed to room temperature naturally
8~10 hours (overnight). Completion of the reaction by TLC. Water was added,
stirred for 30 minutes at room temperature, standing layer, extracted with
hexane, the organic layers were combined, washed to neutrality with saturated
sodium bicarbonate, saturated brine, dried over anhydrous sodium sulfate,
filtered and the filtrate was concentrated under reduced pressure to give 4
dark oil.
e) Prepared at room temperature for 4 to 5,
methanol, water and triethylamine were added to the reaction vessel, stir.
Warmed to 75-80 °C, the reaction 4~5 hours, the reaction was complete, the
system was cooled to below 10 °C, potassium carbonate was added, stirred for 30
minutes, was slowly added sodium borohydride (note the paste), slowly raised It
was stirred at room temperature for 8-10 hours to complete the reaction. At
this time, the reaction system PH = 9-10. Water was added to the system and
quench the reaction, stirred for 0.5 hours, the system was adjusted with
concentrated hydrochloric acid to PH = 2-3, and extracted with ethylacetate,
the organic phase was separated, washed with saturated brine, and the combined
organic phases are dried over anhydrous sodium sulfate, filtered, and
concentrated to give a viscous black 5.
f) Preparation 6: 5, respectively, and absolute ethanol was added L-amino
purified autoclave, stirring chamber for about 1 hour, the crystal
precipitation temperature was raised to 50-60°C, stirred for 5-6 hours, cooled
to room temperature stirred for about 3 hours, filtered, the filter cake was
washed with small amount of absolute ethanol, the filter cake was dried under
reduced pressure at 55°C for 10 hours to give 5 as a light brown powdery solid.
(HPLC San 93%).
g) Preparation 7: addition of methanol at room temperature and at 6, to
ice-salt bath and dry reaction vessel, was added dropwise concentrated sulfuric
acid, controlling the temperature below 5°C. After the addition, naturally
warmed to room temperature (about 20-30°C), reaction was stirred for 10 hours.
TLC monitored the reaction. After completion of the reaction, methanol was
distilled off under reduced pressure and temperature (process, strict control
of the degree of vacuum and temperature, faster), After evaporation to dryness,
cooled to room temperature. After addition of ethyl acetate and water, stirred
for 5 minutes, and extracted with a separatory funnel, the lower water layer
extracted with ethyl acetate, (aqueous layer was put to close the wastewater
collection tank) The organic layers were combined, washed with saturated
aqueous sodium bicarbonate solution to adjust PH to 8-9. If the emulsion was
filtered with a Buchner funnel, (add a layer of celite on the funnel), and the
filtrate fraction with water to a separatory funnel, the upper organic phase
was washed twice with saturated brine, dried over anhydrous sodium sulfate,
filtered solid sodium sulfate was removed. The organic phase was concentrated
under reduced pressure and a temperature of <50°C, to give black product was
7.
h) Preparation 8: 7 at room temperature, methanol and water were added to the
reaction vessel, stir. Warmed to 75-80°C, 3 to 4 hours of reaction, the
reaction was complete, the system was cooled to below 10°C, slowly adding a
reducing agent was slowly warmed to room temperature stirred for 6~7 hours to
complete the reaction. Water was added to the system, the reaction was
quenched, stirred for 0.5 hours, the system was adjusted with concentrated
hydrochloric acid to PH = 5~6, extracted with ethylacetate, the organic phase
was separated, washed with water, brine, combined organic phases were dried
over anhydrous over sodium sulfate, filtered, and concentrated to give a
viscous reddish 8.
i) Preparation 9 are prepared under nitrogen,
was added to the reaction vessel and dried in glacial acetic acid and 8,
stirred and dissolved, was added boron trifluoride acetic acid, heating up the
reaction 5~15 hours, the solution turned black, the reaction completion, was
cooled to room temperature. Methanol was added, the ice bath was added to the
reaction flask with 5N potassium hydroxide solution adjusted to PH 7~9, in this
case yellow emulsion was then slowly added dropwise 30% hydrogen peroxide
solution, the addition, the ice bath was removed, the under nitrogen, heated up
to 70°C incubation for 12 hours. After completion of the reaction, cooled to
room temperature, the batch dropwise addition of saturated sodium bisulfite
solution. After the addition was stirred for half an hour, then the temperature
of the methanol under reduced pressure to recover about 60°C (note the paste,
distillation rate is not too fast, methanol and concentrated after a large
number of foam generator) the residue was cooled to room temperature, cooled
with an ice-salt bath to 0°C, the mixture was adjusted to PH 2 with
concentrated hydrochloric acid, as a yellow liquid. Ethyl acetate was added to
the reaction flask, stirred for 5 minutes, extracted with ethyl acetate
(recyclable apply), if the emulsion serious, can add a little acetone, and the
organic layer was dried over anhydrous sodium sulfate, and concentrated to
obtain a pale yellow oily liquid 9.
j) 9 ketal of Preparation 10 to give 10 is
formed under the action of a ketone.
k) Prepared at room temperature was added
sequentially 3A molecular sieves ll dry dichloromethane and dried to a reaction
vessel, under nitrogen, the reaction solution was cooled to about -25 °C, was
added dropwise (_) - DIPT, dropwise , stirred for 20 minutes at the reaction
temperature of about -25 °C. Then a solution of Ti (i-oft04, dropwise, stirred
at the reaction temperature of about -25 °C 20 min; 10 and then added dropwise
a mixed solution of dichloromethane, dropwise, the reaction was stirred at
temperature of about _25 °C 20 minutes; TBHP solution was then added dropwise,
dropwise, stirring was continued at temperature of about _25 °C the reaction,
the reaction is monitored by TLC developing solvent = petroleum ether: ethyl
acetate = 2:1 (about 4 hours the end of the reaction, this. reaction requires
strictly anhydrous, or incomplete reaction) after completion of the reaction,
aqueous sodium bisulfate was added dropwise, while the internal temperature
does not exceed -10 °C, addition was complete, remove the cooling bath was
allowed to warm to room temperature Q0-25 °C ) The reaction was stirred for 1
hour. Filtration, (add a layer of celite on the funnel), and the filtrate was
added water and, after stirring uniformly vibrating, extracted, the aqueous
layer extracted twice with dichloromethane top organic phases are combined,
washed successively with saturated aqueous sodium bicarbonate and saturated
brine washed twice with water, the organic phase is extracted, dried over
anhydrous sodium sulfate, filtered to remove solid sodium sulfate. Under
reduced pressure to 45<°C temperature organic phase was concentrated to give
25g of a pale brown oil 11.
l) Preparation of 1, 12: successively added at room temperature to the kettle
amino-6-benzyloxy guanine, lithium hydroxide monohydrate of DMF and, under
nitrogen protection, was heated to 90°C, reaction was stirred for about 16
hours the end of the reaction. TLC monitored the reaction. Developing solvent =
petroleum ether: ethyl acetate = 2: 3. After addition of ethyl acetate and
saturated brine, stirred for 5 minutes, filtered (diatomaceous add a layer on
the funnel) the filtrate was transferred to a separatory funnel and extracted
the aqueous layer extracted with ethyl acetate three times below, (the aqueous
layer The organic layers were combined waste water collection tank placed close),
the upper organic phase was successively washed twice with 50% saturated
aqueous citric acid, washed twice with saturated brine, the upper organic phase
was dried over anhydrous sodium sulfate, filtered to remove solids sodium.
Concentration of the organic temperatures <65 °C, to give brown gum, a crude
product 12. Was used directly in the next reaction.
m) At room temperature for 13 preparation was
added to the kettle in dry dichloromethane, into nitrogen, start stirring. 12
was added, the whole solution was heated to micro (about 30-32 °C). Under
nitrogen, at ice-salt bath was added pyridinium p-toluenesulfonate (PPTS),
stirring for 5 minutes the addition was complete, the temperature controlled at
0 °C, was added dropwise triethyl orthoformate, addition was complete, the ice
bath removed the reaction was stirred for 3 hours with warm water temperature
controlled at 25 °C. The reaction was monitored by TLC, developing solvent =
petroleum ether: ethyl acetate = 2:1. After completion of the reaction, saturated
sodium carbonate solution was slowly added, addition was complete the mixture
was stirred at 20-25 °C for half an hour, then added to a separatory funnel and
the stationary layers were separated and the lower organic phase was separated;
the upper aqueous phase, then with dichloromethane extraction time, the upper
aqueous phase into the collection tank.
The combined lower organic phases were then
washed twice with water, the lower organic phase was dried over anhydrous
sodium sulfate, and sodium sulfate to remove solids. The organic phase was
concentrated to a temperature ^65 °C, and then evacuated for 2 hours to give a
viscous oil. Under nitrogen above oil was transferred to a 500ml four-necked
flask, stirred and dissolved after acetic anhydride was added, together with
acetic acid, an antioxidant (BHT) I grains, then dried over anhydrous
oxygen-free state, the incubation was heated to 118-122 °C for about 30 hours.
In the course of the reaction incubation, the solution from dark brown to
black, TLC the reaction was monitored, after the completion of the reaction,
are graded cool. Under nitrogen, when the temperature dropped to 65 °C, diluted
with water and ethyl acetate were added, the ethyl acetate layer separated,
washed with brine, dried over anhydrous sodium sulfate, pouring suspended
solids, concentrated, the solid washed with cold ethyl acetate, dried 13 as a
white solid. Preparation of [eta], entecavir monohydrate 13, was added 1: 1 THF
- methanol, hydrochloric acid was added dropwise 2Ν The reaction was stirred
until the starting material was 4.5h at 60 °C, cooled to room temperature,
diluted with water and ethyl acetate were added, with vigorous stirring PH was
adjusted with iN sodium hydroxide to 7.0, was allowed to stand, a white solid
in the organic layer, two phases were separated, the aqueous phase was
extracted with ethyl acetate, the combined organic phases were washed with
brine, dried over anhydrous sodium sulfate, pouring suspended solids,
concentrated solid was washed with cold ethyl acetate, and dried to give
entecavir monohydrate as a white solid (Figure 4).
As shown in Scheme 1, compound 3 was prepared as
a single diastereomer from 3kg of 92% ee (S)-(+)-carvone via a twostep
transformation including a stereoselective epoxidation and chlorohydrin
formation from the newly formed epoxide. Tosylation of the sec-hydroxyl group
of compound 3 afforded 4.25 kg of product 4 (60% yield over 3 steps) in 100% ee
after recrystallization from MeOH. This ultra-pure intermediate was then
reacted with mCPBA to afford epoxide 5, which was converted into diol 6 after
treatment with dilute aqueous sulfuric acid. Protection of the diol with
dimethoxypropane afforded 3.4 kg of intermediate 7 (67% over 3 steps).This
product was treated with sodium methoxide in methanol to initially provide the
cis-substituted Favorskii rearrangement product 8a, which upon isomerization
gave the thermodynamically more stable cyclopentanecarboxylate 8 under the
reaction conditions, though the epimerization was incomplete even after being
stirred for 24 hours (50 g scale) at room temperature. Fortunately, the problem
was solved by using methyl t-butyl ether (MTBE)/methanol as the solvent and the
reaction was complete in less than 17 hours (50g scale).
Molecular Modeling Study of Drug-Resistant HBV
Molecular dynamics studies on the homology model
structure of HBV can provide useful information regarding mutations associated
with resistance to inhibitors of HBV polymerase. Daga et al. [8] built a
stereochemically significant homology model of HBV polymerase and suggested a
significant role for conserved Lys 32 residue in HBV RT, which corresponds to
Lys 65 in HIV RT, in binding of nucleotides and known HBV RT inhibitors. Their
homology model of HBV polymerase had two main differences from previous
reports: They aligned the sequence by using the proper match of a conserved Lys
residue in HIV-1 RT, which has important salt bridge interactions with the
γ-phosphate of the incoming nucleotide.
Secondly, they used a different template structure
of HIV-1 RT (PDB code: 1T05) that was a higher resolution crystal structure
compared to the previously. sed template (PDB code: 1RTD). Based on this
modeling result, they provided an explanation for the various resistant mutants
of HBV polymerase and successfully predicted binding conformations of known HBV
inhibitors [8]. Das K et al. [2] constructed a three-dimensional homology model
of the catalytic core of HBV polymerase based on the crystal structure of HIV-1
RT. Molecular modeling studies using the HBV polymerase homology model suggest
that steric hindrance between the mutant amino acid side chain and lamivudine
or emtricitabine, anti-HBVdrug, could account for the resistance phenotype.
Specifically, steric conflict between the Ile or Val at position rt204 in HBV
polymerase and the sulfur atom in the oxathiolane ring of lamivudine and
emtricitabine is proposed to account for the resistance observed with rtM204I
or rtM204V mutation. The effects of the rtL180M mutation, which also occurs
near the HBV polymerase active site, appeared to be less direct, potentially
involving rearrangement of the deoxynucleoside triphosphate-binding pocket
residues. Sharon [5] constructed a homology model structure of HBV polymerase,
which is used for minimization, conformational search and induced fit docking
followed by binding energy calculation for wild-type and mutant HBV polymerases
(rtL180M, rtM204V, rtM204I, rtL180M + rtM204V, rtL180M + rtM204I). Their
studies suggest a significant correlation between the fold resistance and the
binding affinity of five anti-HBV agents: lamivudine, adefovir, entecavir,
telbivudine and clevudine. Also, they analyzed different binding modes for
synthetic nucleoside analogue drugs as well as natural nucleosides.
Although their studies may not fully explain the
difference of quantitative binding affinity, they showed detailed resistance
mechanisms for anti-HBV drugs against wild-type and mutant HBV. Adefovir is
active against wild-type and lamivudine-resistant strains of HBV [9]. In contrast
to lamivudine therapy, adefovir is associated with delayed and uncommon
selection of drug-resistant viruses [10]. Long-term treatment with adefovir
dipivoxil leads to the rtN236T mutation, which displays reduced susceptibility
to adefovir but remains sensitive to lamivudine [11]. Yadav V et al. [12]
presented the molecular basis of the mechanism of adefovirdiphosphate against
lamivudineresistant mutants and its decrease in susceptibility for rtN236T HBV
polymerase mutants. These molecular dynamics studies demonstrated that the
rtN236T HBV polymerase mutation does not affect the binding affinity of the
natural substrate (dATP), but it decreases the binding affinity of
adefovirdiphosphate toward the rtN236T HBV polymerase.
The lamivudine-resistant mutations, rtM204V and
rtM204I, result in increased vanderwaals contacts between adefovirdiphosphate
and the mutated residues, which accounts for the better binding affinity of
adefovir-diphosphate toward these mutants. The second lamivudine-related
mutation, rtL180M, also results in increased van der Waals interactions between
adefovirdiphosphate and the final residue of the primer strand, which accounts
for the better binding affinity of adefovir-diphosphate in these mutants.
Warner et al. [1] determined the prevalence of rtL80V/I mutation in
lamivudine-resistant HBV isolates and characterized the in vitro phenotype of
the mutants. Although L80I increases sensitivity to lamivudine and imparts a
replication defect, it enhances the in vitro replication of lamivudine-resistant
(rtM204I) HBV.
Molecular modeling revealed that Leu 80 does not
interact directly with the enzyme’s substrates. Molecular models of HBV reverse
transcriptase showed that, although Leu 80 is located distal to the enzyme’s
dNTP binding pocket, substitution of isoleucine for leucine at this site
partially restores replication efficiency by sufficiently changing the overall
spatial alignment of other residues that are important for catalysis. These
results imply that the presence of rtL80I decreases the enzyme’s, affinity for
both dNTPs and lamivudine triphosphate and that the decrease in affinity for
lamivudine triphosphate is greater than the decrease in affinity for the
natural substrate, dCTP. As mentioned above, using the homology model structure
of HBV polymerase, the amino acid changes resulting from mutations that give
antiviral resistance can be mapped to functional regions to provide a better
understanding of the molecular mechanism of resistance [2,6]. The HBV
polymerase consists of four different domains: terminal protein, a space
region, a catalytic RT domain and RNase H domain. We constructed and refined
the model structure of HBV RT based on the homology to HIV-1 RT according to
the reported method [8] (Figure 1). The ribbon diagram of homology model
structure of HBV RT shows the location of the major mutations that confer
resistance to clinically available six drugs. The HBV RT model structure was
constructed and refined as previously reported [8]. HBV RT consists of three
sub-domains: fingers (amino acid 1 to 49 and 90 to 172, in green), palm (amino
acid 50 to 89 and 173 to 267, in cyan), and thumb (amino acid 268 to 351, in
yellow). The locations of the mutations are indicated with the sphere model
(Figures 5 & 6).
Conclusion
Treatment for chronic hepatitis B patients
depends on anti-HBV drugs. Even though the currently approved anti-HBV drugs,
nucleos(t)ide analogues, show potent and fast antiviral response, several
problems remain to be solved. These include the development of resistance and
the side effects such as myopathy, which is induced by mitochondrial damage.
Based on advances in the development of antiviral agents along with newly
discovered drug candidates and combination therapy, resistance should not be a
great concern in the near future. However, combination therapy for effective
control of HBV requires the development of novel drugs that have different
mechanisms of action. The mitochondrial damage is mainly due to the high
affinity of nucleos(t)ide RT inhibitors for mitochondrial DNA polymerase gamma.
Therefore, alleviation of side effects should be
considered in the development of future nucleos (t)ide drugs. In this regard,
the fine crystal structure of polymerase gamma was reported recently [13,14].
The elucidation of the polymerase gamma structure establishes a foundation for
understanding the molecular basis of the toxicity of anti-retroviral drugs
targeting HBV and HIV and the cause of cellular toxicity induced by some
antiviral nucleoside analogs [15-132]. Eventually, these fundamental studies in
conjunction with advanced drug development tools will provide valuable
information for the development of novel drugs without side effects. New
process of Entecavir well defined in patent EP2488522
https://lupinepublishers.com/food-and-nutri-journal/pdf/SJFN.MS.ID.000114.pdf
https://lupinepublishers.com/food-and-nutri-journal/fulltext/entecavir-patent-evaluation-molecular-modelling-study-of-drug-resistant-hbv.ID.000114.php
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