2. D. J. S.Newman, thesis, University of Exeter (1982);
`A. R. Ennos, J. Exp. Biol. 142, 87 (1989).
`3. S. Dalton, Borne on the Wind (Chatto & Windus,
`London, 1975); G. Ru¨ppell, J. Exp. Biol. 144, 13
`(1989); J. H. Brackenbury, Insects in Flight (Bland-
`ford, London, 1992);
`J. M. Wakeling and C. P.
`Ellington, J. Exp. Biol. 200, 557 (1997);
`ibid., p.
`583.
`4. R. Å. Norberg, J. Comp. Physiol. 81, 9 (1972).
`5. R. J. Wootton, Adv. Odonatol. 5, 153 (1991).
`
`6. 㛬㛬㛬㛬 , J. Exp. Biol. 180, 105 (1993).
`
`7. E. F. Riek and J. Kukalova´-Peck, Can. J. Zool. 62, 1150
`(1984).
`8. C. Brauckmann and W. Zessin, Dtsch. Entmol. Z. 36,
`177 (1989).
`
` on May 15, 2015
` on May 15, 2015
` on May 15, 2015
` on May 15, 2015
`
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`www.sciencemag.org
`
`Downloaded from
`Downloaded from
`Downloaded from
`Downloaded from
`
`1 June 1998; accepted 22 September 1998
`
`emia, is caused by mutations in apoB (6, 7).
`Heterozygous people with this disease have
`half the normal
`levels of apoB-containing
`lipoproteins and lack the clinical signs and
`symptoms of patients with abetalipoproteine-
`mia. Family studies have shown that individ-
`uals with hypobetalipoproteinemia have a
`prolonged life span (8).
`We performed a high-throughput screen of a
`large chemical library to identify inhibitors of
`MTP-mediated triglyceride transfer. This re-
`sulted in the discovery of BMS-200150 (com-
`pound 1 in Fig. 1), which inhibits the MTP-
`mediated transport of triglycerides between
`membranes in vitro (9) and inhibits the secre-
`tion of apoB-containing lipoproteins from
`HepG2 cells, a human liver-derived cell line.
`However, the compound was not active in an-
`imal models. Compound 2, an analog of BMS-
`200150 with an extended alkyl linker, was of
`comparable potency in both the lipid transfer
`and HepG2 apoB secretion assays (Fig. 1 and
`Table 1). A fluorenyl amide (compound 3),
`which is a much less potent inhibitor, was also
`identified in the high-throughput screening.
`Subsequently, nitrogen substitution of 3 with a
`short-chain alkyl group (for example, 4) was
`
`J. R. Wetterau, R. E. Gregg, T. W. Harrity, C. Arbeeny, M.
`Cap, F. Connolly, C.-H. Chu, R. J. George, D. A. Gordon, H.
`Jamil, K. G. Jolibois, L. K. Kunselman, T. J. Maccagnan, B.
`Ricci, M. Yan, D. Young, Department of Metabolic Dis-
`eases, Bristol-Myers Squibb Pharmaceutical Research In-
`stitute, Princeton, NJ 08543–4000, USA. S.-J. Lan, De-
`partment of Metabolism and Pharmacokinetics, Bristol-
`Myers Squibb Pharmaceutical Research Institute, Prince-
`ton, NJ 08543–4000, USA. Y. Chen, O. M. Fryszman,
`J. V. H. Logan, C. L. Musial, M. A. Poss, J. A. Robl, L. M.
`Simpkins, W. A. Slusarchyk, R. Sulsky, P. Taunk, D. R.
`Magnin, J. A. Tino, R. M. Lawrence, J. K. Dickson Jr., S. A.
`Biller, Division of Discovery Chemistry, Bristol-Myers
`Squibb Pharmaceutical Research Institute, Princeton, NJ
`08543–4000, USA.
`*To whom correspondence should be addressed. E-
`mail: Wetterau_John_R@msmail.bms.com (J.R.W.)
`and biller@bms.com (S.A.B.)
`†Present address: GelTex Pharmacueticals, 9 Fourth
`Avenue, Waltham, MA 02154, USA.
`‡Present address: 14 Royal College of Surgeons, Dub-
`lin, Ireland.
`§Present address: Department of Chemistry, 516
`Physical Sciences 1, University of California, Irvine, CA
`92697, USA.
`
`on which they feed. Carboniferous odonatoids
`were already predatory (8), but no contempo-
`rary insects would have approached the maneu-
`verability of many extant species. The relatively
`smaller, less sturdy bodies and consequent low-
`er wing-loadings of Eugeropteridae indicate
`lower maximum speeds than in Anisoptera, and
`the absence of a nodus suggests a poorer capa-
`bility for wing twisting, truncating the lower
`end of their speed range: It is unlikely that they
`could hover like modern dragonflies. Nonethe-
`less, the flexible trailing edge and the shortened
`subcostal vein indicate that some supinatory
`twisting was possible, and the group appears to
`
`R E P O R T S
`
`be following a trend, parallelled in many other
`insect groups, toward improving flight versatil-
`ity by recruiting upstroke forces to supplement
`those of the far more effective downstroke, and
`varying their magnitude and direction at need.
`The “smart” wing-base mechanism is best in-
`terpreted as an elegant means of maintaining
`downstroke efficiency in the presence of these
`adaptations to improve upstroke usefulness.
`
`References
`1. R. J. Wootton, J. Zool. London 193, 447 (1981); Sci.
`Am. 263, 114 (November 1990); Annu. Rev. Entomol.
`37, 113 (1992).
`
`An MTP Inhibitor That
`Normalizes Atherogenic
`Lipoprotein Levels in
`WHHL Rabbits
`
`John R. Wetterau,* Richard E. Gregg, Thomas W. Harrity,
`Cynthia Arbeeny,† Michael Cap, Fergal Connolly,‡
`Ching-Hsuen Chu, Rocco J. George, David A. Gordon, Haris Jamil,
`Kern G. Jolibois, Lori K. Kunselman, Shih-Jung Lan,
`Thomas J. Maccagnan, Beverly Ricci, Mujing Yan, Douglas Young,
`Ying Chen, Olga M. Fryszman,§ Janette V. H. Logan,
`Christa L. Musial, Michael A. Poss, Jeffrey A. Robl,
`Ligaya M. Simpkins, William A. Slusarchyk, Richard Sulsky,
`Prakash Taunk, David R. Magnin, Joseph A. Tino,
`R. Michael Lawrence, John K. Dickson Jr., Scott A. Biller*
`
`Patients with abetalipoproteinemia, a disease caused by defects in the micro-
`somal triglyceride transfer protein (MTP), do not produce apolipoprotein B–con-
`taining lipoproteins. It was hypothesized that small molecule inhibitors of MTP
`would prevent the assembly and secretion of these atherogenic lipoproteins. To
`test this hypothesis, two compounds identified in a high-throughput screen for
`MTP inhibitors were used to direct the synthesis of a highly potent MTP
`inhibitor. This molecule (compound 9) inhibited the production of lipoprotein
`particles in rodent models and normalized plasma lipoprotein levels in Wa-
`tanabe-heritable hyperlipidemic ( WHHL) rabbits, which are a model for human
`homozygous familial hypercholesterolemia. These results suggest that com-
`pound 9, or derivatives thereof, has potential applications for the therapeutic
`lowering of atherogenic lipoprotein levels in humans.
`
`Apolipoprotein B (apoB)–containing lipopro-
`teins [chylomicrons, very low density li-
`poproteins (VLDL) and their respective met-
`abolic products, chylomicron remnants, and
`low density lipoproteins (LDL)] promote cor-
`onary artery atherosclerosis, which is a lead-
`ing cause of death in industrialized nations.
`MTP is a heterodimeric lipid transfer protein
`consisting of protein disulfide isomerase and
`a unique 97-kD subunit that is localized in the
`endoplasmic reticulum of hepatocytes and
`enterocytes (1–3). Defects in MTP cause abe-
`talipoproteinemia (3–5), a disorder in which
`the production of VLDL and chylomicrons is
`
`disrupted. Patients with abetalipoproteinemia
`have plasma cholesterol levels of ⬃40 mg/dl
`and plasma triglyceride levels of ⬍10 mg/dl
`(6), whereas normal adults have levels of 180
`to 220 and 100 to 150 mg/dl, respectively.
`These findings suggest that inhibitors of MTP
`might be therapeutically useful for inhibiting
`the production of VLDL and chylomicrons,
`thereby reducing the levels of atherogenic
`lipoprotein particles.
`Abetalipoproteinemia is an extreme ex-
`ample of MTP inhibition and would not be
`the intended clinical end point for a drug. A
`related genetic disease, hypobetalipoprotein-
`
`www.sciencemag.org SCIENCE VOL 282 23 OCTOBER 1998
`
`751
`
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`
`found to improve the potency of this series,
`making it comparable with the series of 1 and 2.
`On the basis of the structural relationships
`between 1, 2, and 4, it was proposed that 4
`binds to MTP at a subdomain of the binding
`site for 1 and 2. To test this proposal, we
`prepared “hybrid” analogs 5 and 6 (Fig. 1).
`With 5, the overlap between the fluorenyl
`amide of 4 and the isoindolone of 1 was
`explored, resulting in a significant loss in
`inhibitory potency (Table 1). With 6,
`the
`overlap between the fluorenyl amide of 4 and
`the diphenylmethyl substituent of 2 was ex-
`plored, resulting in a dramatic improvement
`(⬎100-fold) in potency in both the lipid
`transfer and HepG2 apoB secretion assays.
`Hybrid 6 was tested for its ability to in-
`hibit lipoprotein secretion in fasted rats. This
`assay was based on the ability of intravenous-
`ly injected Triton WR1339 to prevent the
`catabolism of triglyceride-rich lipoproteins
`(10). Thus, after the administration of Triton,
`there was a linear increase in plasma triglyc-
`eride levels, which reflects the triglyceride
`secretion rate from the liver and intestine.
`The inhibition of hepatic lipoprotein produc-
`tion can be measured with fasted animals in
`which the liver is the primary source of plas-
`ma triglycerides, whereas in fed rats, there is
`substantial lipoprotein production in the in-
`testine. In contrast
`to the earlier analogs,
`hybrid 6 was a potent and dose-dependent
`inhibitor of triglyceride secretion in vivo. The
`corresponding N-CH2CF3 analog, 7, resulted
`in a dramatic improvement in oral potency.
`A modification of the isoindolone portion of
`7 indicated that a benzamide (inhibitor 8) was a
`suitable replacement for the isoindolone ring.
`An extensive optimization of this substituent
`was performed by an automated organic syn-
`thesis (11), which resulted in the identification
`of the 4⬘-CF3-biphenyl carboxamide, 9. This
`MTP inhibitor possesses subnanomolar poten-
`cy in both the lipid transfer and HepG2 apoB
`secretion assays and inhibits lipoprotein secre-
`tion in fasted rats (Table 1). The intravenous
`and oral half-maximal effective dose (ED50)
`values were similar, suggesting that the com-
`pound was well-absorbed and bioavailable to
`the liver. Compound 9 was equally effective in
`inhibiting acute lipoprotein secretion in fasted
`or fed rats (Fig. 2), indicating that it inhibits
`both hepatic and intestinal lipoprotein secretion.
`The effect of compound 9 on plasma lipid
`levels was tested in hamsters which, unlike
`other rodents, transport a substantial propor-
`tion of their cholesterol on LDL. Hamsters
`that were fed a standard, low-fat, high-carbo-
`hydrate diet were treated once daily with
`doses of compound 9 of 1, 3, or 6 mg per
`kilogram of body weight (mg/kg) for 7 days.
`After the final drug treatment, the animals
`were fasted for 18 hours, and their plasma
`lipid and lipoprotein levels were then evalu-
`ated (Fig. 3). A dose-dependent decrease in
`
`R E P O R T S
`
`the total plasma cholesterol was observed,
`with an ED50 value for cholesterol lowering
`of 2.4 mg/kg. The triglyceride, VLDL and
`LDL cholesterol, and high density lipoprotein
`(HDL) cholesterol levels all decreased in par-
`allel with the decrease in total plasma choles-
`terol. The HDL decrease was unlikely to be
`due to a direct effect on HDL production
`because, in HepG2 cells, the ED50 for the
`inhibition of secretion of apolipoprotein AI
`(apoAI; the major structural protein of HDL)
`by compound 9 is over 8000-fold higher than
`that for the inhibition of secretion of apoB
`(Table 1). At the 6 mg/kg dose, the total
`plasma cholesterol and triglyceride levels
`were decreased by 90 and 49%, respectively,
`in comparison to the control animals. When
`animals were treated with compound 9 for up
`to 3 weeks, there were similar findings. There
`were no clinically relevant differences be-
`tween vehicle- and drug-treated animals in
`body weight, plasma aspartate aminotransfer-
`ase (AST), alanine aminotransferase (ALT),
`or any other plasma chemistry variables that
`were tested (12).
`Hamsters that were treated with com-
`pound 9 did not have steatorrhea, as indicated
`by the absence of fat (⬍0.01 g per 24 hours)
`in their stools. After killing these hamsters,
`we noted visual and biochemical evidence of
`fat accumulation in enterocytes. The liver
`triglyceride content of animals that were
`treated with compound 9 reached a plateau
`after 1 week of treatment, never exceeded 25
`mg per gram of tissue (13), and returned to
`control levels 48 hours after the termination
`of a 3-week treatment regimen. In compari-
`son, fatty livers in humans contain up to 35%
`triglyceride by weight (14). Liver weight in
`the hamsters
`(as percent body weight)
`showed minimal change with treatment (15).
`Plasma transaminase levels did not rise sig-
`
`Fig. 1. Structures of MTP inhibitors.
`
`Table 1. Comparative activity of compounds as MTP inhibitors measured in vitro, in cell culture, and in
`vivo. The in vitro and cell culture assays were performed as described in (9). The fasted rat assay was
`performed as described in Fig. 2 and (10). For intravenous (iv) dosing, the compound was directly
`incorporated into Triton WR1339. Human MTP was produced in Sf9 insect cells with the baculovirus
`expression system (4).
`IC50, concentration that produces half-maximal
`inhibition; ED50, dose that
`produces a half-maximal effect; IA@2, IA@15, and IA@100, inactive at doses 2, 15, and 100 mg/kg,
`respectively; ND, not done.
`
`Compound
`
`IC50 values of
`triglyceride transfer
`of human MTP in
`vitro (nM)
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`
`2,200
`5,800
`36,000
`2,200
`25,300
`12
`36
`23
`0.5
`
`ED50 values of secretion
`of apoB and apoAI from
`HepG2 cells (nM)
`
`ED50 values of
`secretion of
`triglycerides in rats
`(mg/kg)
`
`apoB
`
`1,800
`1,100
`17,000
`4,500
`2,600
`19
`3
`8.1
`0.8
`
`apoAI
`
`⬎30,000
`11,000
`⬎33,000
`⬎33,000
`4,600
`⬎3,300
`⬎3,300
`68,000
`6,500
`
`iv
`
`IA@2
`ND
`ND
`ND
`ND
`0.28
`1.2
`ND
`0.15
`
`Oral
`
`IA@15
`ND
`ND
`IA@100
`ND
`15
`1.9
`0.7
`0.19
`
`752
`
`23 OCTOBER 1998 VOL 282 SCIENCE www.sciencemag.org
`
`CFAD Ex. 1018 (2 of 5)
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`

`
`R E P O R T S
`
`Fig. 2. Inhibition by compound 9
`of triglyceride secretion in fasted
`and fed rats. Sprague-Dawley
`four per
`rats (⬃200 g each,
`treatment group) were adapted
`to a reversed diurnal light cycle
`for 2 weeks. Before the experi-
`ment, the rats either were fasted
`(solid bars) or had free access to
`food (hatched bars) for 18 hours.
`The animals were orally dosed
`with compound 9 in a vehicle
`(10% M-pyrol, 80% water, 5%
`cremophore, and 5% ethanol) 1
`hour before receiving an intrave-
`nous injection of Triton WR1339
`(250 mg/kg). The triglyceride se-
`cretion rate was determined by
`calculating the amount of tri-
`glyceride that was accumulated
`in plasma during the 2.5 hours
`after the Triton injection. The
`standard assay was linear for at least 5 hours after the Triton injection. Plasma triglyceride levels
`were determined with a Roche Cobas blood chemistry autoanalyzer (a negative percentage
`indicates a decrease). The animals were cared for in accordance with institutional guidelines. Error
`bars indicate SEM. **, significantly different from vehicle-treated animals at P ⬍ 0.005.
`
`Fig. 3. Effect of compound 9 on
`(A) total cholesterol, (B) triglyc-
`erides, (C) VLDL and LDL choles-
`terol, and (D) HDL cholesterol
`levels in hamsters (a negative
`percentage indicates a decrease).
`Male Golden Syrian hamsters
`(⬃140 g each, four per treat-
`ment group) were adapted to
`and maintained on a reverse di-
`urnal
`light cycle. They were
`dosed orally once a day with
`compound 9 in a vehicle (10%
`M-pyrol, 80% water, 5% cremo-
`phore, and 5% ethanol) and were
`allowed free access to a standard
`hamster diet (Purina 5001, which
`contains 0.02% cholesterol and
`4.5% triglyceride). After 7 days
`of drug treatment, hamsters
`were fasted for 18 hours, after
`which plasma lipid levels and
`chemistries were determined with a Roche Cobas blood chemistry autoanalyzer. Hamster lipopro-
`tein fractions were quantitated after the precipitation of apoB-containing lipoproteins with
`phosphotungstate and magnesium chloride. Drug effects were calculated in relation to the vehicle
`control group. Error bars indicate SEM.*, P ⬍ 0.05; **, P ⬍ 0.005.
`
`nificantly over a 3-week treatment period,
`and triglyceride accumulation was reversible,
`which suggest that the modest increase in
`hepatic lipid had no adverse effects in the
`hamster model.
`We also investigated the effect of a
`2-week treatment with compound 9 in ho-
`mozygous Watanabe-heritable hyperlipid-
`emic (WHHL) rabbits whose hepatic LDL
`receptor activity is ⬍5% that of normal rab-
`bits, resulting in dramatically elevated levels
`of apoB-containing lipoproteins. WHHL rab-
`bits are a model for human homozygous fa-
`milial hypercholesterolemia (FH) (16). The
`elevated plasma cholesterol levels in FH pa-
`tients (600 to 1200 mg/dl) cannot be normal-
`ized with current therapies, which typically
`
`lower LDL cholesterol levels by a maximum
`of 30%. The ED50 value for cholesterol low-
`ering in WHHL rabbits that were treated with
`compound 9 was 1.9 mg/kg. Triglyceride lev-
`els were lowered in parallel. At a dose of 10
`mg/kg of 9, the plasma levels of atherogenic,
`apoB-containing, lipoprotein particles were
`essentially normalized (Fig. 4) with no alter-
`ation in plasma AST or ALT (17).
`In the search for an understanding of li-
`poprotein metabolism and a means to treat ath-
`erosclerosis, much effort has been focused on
`hyperlipidemic states. This focus has resulted in
`the identification of genetic defects that produce
`loss of functions that cause hyperlipidemia. Our
`results highlight the importance of understand-
`ing hypolipidemic states. Identifying a loss-of-
`
`Fig. 4. Effect of compound 9 on plasma lipid
`levels in WHHL rabbits (a negative percentage
`indicates a decrease). Five rabbits were treated
`orally for 14 days with compound 9 (10 mg/
`kg). Plasma (A) total cholesterol and (B) triglyc-
`eride levels were measured 18 hours after the
`last dose. Prebleed, solid bars; 14-day treat-
`ment, hatched bars.
`
`function gene defect for a genetic disorder that
`is the physiological opposite of the disease that
`one is attempting to treat allows one to identify
`a target that is theoretically amenable to phar-
`macological intervention (that is, a normal pro-
`tein for which an inhibitor mimicking the effect
`of the mutation will produce the desired thera-
`peutic outcome). With current technologies, it
`is easier to identify a small molecule inhibitor
`of a normal activity than to augment a deficient
`activity. This strategy yielded compound 9,
`which has the potential to be an effective anti-
`atherogenic agent and is now in clinical trials.
`Similar drug discovery strategies may be appli-
`cable to other diseases.
`
`References and Notes
`1. J. R. Wetterau and D. B. Zilversmit, Biochim. Biophys.
`Acta 875, 610 (1986).
`2. J. R. Wetterau, K. A. Combs, S. N. Spinner, B. J. Joiner,
`J. Biol. Chem. 265, 9800 (1990).
`3. D. Sharp et al., Nature 365, 65 (1993).
`4. B. Ricci et al., J. Biol. Chem. 270, 14281 (1995).
`5. E. F. Rehberg et al., ibid. 271, 29945 (1996); C. C.
`Shoulders et al., Hum. Mol. Genet. 2, 2109 (1993);
`T. M. E. Narcisi et al., Am. J. Hum. Genet. 57, 1298
`(1995).
`in The Metabolic and
`6. J. P. Kane and R. J. Havel,
`Molecular Bases of Inherited Disease, C. R. Scriver,
`A. L. Beaudet, W. S. Sly, D. Valle, Eds. (McGraw-Hill,
`
`www.sciencemag.org SCIENCE VOL 282 23 OCTOBER 1998
`
`753
`
`CFAD Ex. 1018 (3 of 5)
`
`

`
`R E P O R T S
`
`New York, ed. 7, 1995), pp. 1853–1885. Abetali-
`poproteinemia is an autosomal recessive disease in
`which plasma VLDL and LDL are virtually absent.
`Affected people have fat malabsorption and have
`triglyceride accumulation in enterocytes and the liv-
`er. Secondary to a vitamin E deficiency, affected
`people may have spinocerebellar ataxia, peripheral
`neuropathy, degenerative pigmentary retinopathy,
`and ceroid myopathy.
`7. M. F. Linton, R. V. Farese Jr., S. G. Young, J. Lipid Res.
`34, 521 (1993).
`8. C. J. Glueck, P. S. Gartside, M. J. Mellies, P. M. Steiner,
`Trans. Assoc. Am. Physicians 90, 184 (1977).
`9. H. Jamil et al., Proc. Natl. Acad. Sci. U.S.A. 93, 11991
`(1996).
`10. R. Borensztajn, M. Rone, T. Kotlar, Biochem. J. 156,
`539 (1976).
`11. R. M. Lawrence, S. A. Biller, O. M. Fryszman, M. A.
`Poss, Synthesis 1997, 553 (1997).
`
`12. Plasma chemistries for treatments with vehicle and 1,
`3, and 6 mg/kg doses of compound 9 were as follows:
`ALT (U/liter), 73 ⫾ 3 (mean ⫾ SEM),65 ⫾ 5, 75 ⫾ 5,
`and 71 ⫾ 2, respectively; AST (U/liter), 58 ⫾ 2, 45 ⫾
`3, 58 ⫾ 2, and 59 ⫾ 3; alkaline phosphatase (U/liter),
`131 ⫾ 11, 137 ⫾ 1, 135 ⫾ 1, and 131 ⫾ 3; lactic
`dehydrogenase (U/liter), 156 ⫾ 5, 136 ⫾ 3, 150 ⫾ 3,
`and 139 ⫾ 5; amylase (U/liter), 1400 ⫾ 300, 1600 ⫾
`100, 1500 ⫾ 200, and 1700 ⫾ 500; gamma glu-
`tamyltransferase (U/liter), 2.3 ⫾ 0.7, 2.3 ⫾ 0.2, 1.8 ⫾
`0.4, and 2.2 ⫾ 0.5; glucose (mg/dl), 230 ⫾ 7, 235 ⫾
`8, 194 ⫾ 22, and 199 ⫾ 15; creatinine (mg/dl), 0.4 ⫾
`0, 0.4 ⫾ 0, 0.4 ⫾ 0, and 0.4 ⫾ 0; blood urea nitrogen
`(mg/dl), 14 ⫾ 1, 14 ⫾ 1, 15 ⫾ 1, and 18 ⫾ 2; total
`protein (g/dl), 5.4 ⫾ 0.1, 5.5 ⫾ 0.1, 5.7 ⫾ 0.2, and
`5.2 ⫾ 0.5; and albumin (g/dl), 3.7 ⫾ 0.1, 3.6 ⫾ 0.1,
`3.6 ⫾ 0.1, and 3.6 ⫾ 0.1.
`13. The milligrams of triglyceride measured per gram of
`wet weight liver for treatments with vehicle and 1, 3,
`
`and 6 mg/kg doses of compound 9 were as follows:
`9.1 ⫾ 0.6 (mean ⫾ SEM), 11 ⫾ 0.3, 18 ⫾ 1.8, and
`24 ⫾ 0.4, respectively, at 1 week; 8 ⫾ 0.4, 11 ⫾ 1.1,
`12 ⫾ 0.8, and 19 ⫾ 3.4 at 2 weeks; and 9 ⫾ 0.5, 11 ⫾
`1.5, 23 ⫾ 3.5, and 19 ⫾ 1.0 at 3 weeks.
`14. S. R. Cairns and T. J. Peters, Clin. Sci. 65, 645 (1983).
`15.
`In an independent 3-week study, liver weight mea-
`sured as percent of body weight was 11% higher in
`hamsters that were treated with a 6 mg/kg dose of
`compound 9 (4.1 ⫾ 0, mean ⫾ SEM) than in vehicle-
`treated hamsters (3.7 ⫾ 0.1).
`16. T. Kita, M. S. Brown, Y. Watanabe, J. L. Goldstein,
`Proc. Natl. Acad. Sci. U.S.A. 78, 2268 (1981).
`17. The ALT and AST levels of drug-treated rabbits were
`54.0 ⫾ 7.4 and 34.6 ⫾ 3.5 U/liter (mean ⫾ SEM) in
`comparison to vehicle-treated rabbit levels of 48.3 ⫾
`5.2 and 28.8 ⫾ 12.5 U/liter.
`
`18 June 1998; accepted 23 September 1998
`
`Genome Sequence of an
`Obligate Intracellular Pathogen
`of Humans: Chlamydia
`trachomatis
`
`Richard S. Stephens,* Sue Kalman, Claudia Lammel, Jun Fan,
`Rekha Marathe, L. Aravind, Wayne Mitchell, Lynn Olinger,
`Roman L. Tatusov, Qixun Zhao, Eugene V. Koonin,
`Ronald W. Davis
`
`Analysis of the 1,042,519 – base pair Chlamydia trachomatis genome revealed
`unexpected features related to the complex biology of chlamydiae. Although
`chlamydiae lack many biosynthetic capabilities, they retain functions for per-
`forming key steps and interconversions of metabolites obtained from their
`mammalian host cells. Numerous potential virulence-associated proteins also
`were characterized. Several eukaryotic chromatin-associated domain proteins
`were identified, suggesting a eukaryotic-like mechanism for chlamydial nucle-
`oid condensation and decondensation. The phylogenetic mosaic of chlamydial
`genes, including a large number of genes with phylogenetic origins from eu-
`karyotes, implies a complex evolution for adaptation to obligate intracellular
`parasitism.
`
`Chlamydia are bacterial pathogens whose
`representatives are widely distributed in na-
`ture, and C. trachomatis causes several hu-
`man diseases of medical significance. Ocular
`infection leads to trachoma, a leading cause
`of preventable blindness. Of all infectious
`diseases reported to U.S. state health depart-
`
`R. S. Stephens, C. Lammel, W. Mitchell are with the
`Program in Infectious Diseases, University of Califor-
`nia, Berkeley, CA 94720, USA, and the Francis I.
`Proctor Foundation, University of California, San
`Francisco, CA 94143, USA. S. Kalman, J. Fan, R. Mar-
`athe, R. W. Davis are at the DNA Sequencing and
`Technology Center, Stanford University, Stanford, CA
`94305, USA. L. Aravind, R. L. Tatusov, E. V. Koonin are
`at the National Center for Biotechnology Information,
`National Library of Medicine, National Institutes of
`Health, Bethesda, MD 20894, USA. L. Olinger and Q.
`Zhao are with the Francis I. Proctor Foundation, Uni-
`versity of California, San Francisco, CA 94143, USA.
`*To whom correspondence should be addressed. E-
`mail: ctgenome@socrates.berkeley.edu
`
`ments and the U.S. Centers for Disease Con-
`trol and Prevention, chlamydial genital tract
`infections are the most common, and infec-
`tion of the genital tract often results in pelvic
`inflammatory disease, ectopic pregnancy,
`chronic pelvic pain, epididymitis, and infant
`pneumoniae (1). Chlamydia trachomatis gen-
`ital
`tract
`infections may also significantly
`increase the risk for HIV infection (2).
`Chlamydiae are deeply separated from other
`eubacteria and represent one of the kingdom-level
`branches of the phylogenetic tree (3). After inva-
`sion of eukaryotic cells, chlamydiae grow within
`an intracellular vacuole, called an inclusion, that
`does not fuse with lysosomes. Microbiological-
`ly, Chlamydia are characterized by a develop-
`mental cycle involving a metabolically inac-
`tive infectious developmental form called the
`elementary body (EB) that, after entry into
`the target host cell, differentiates into a met-
`abolically active developmental form called
`
`the reticulate body (RB). At approximately 20
`hours post infection and after multiple divi-
`sions by binary fission, the RB differentiates
`into the EB developmental stage and infec-
`tious EBs are released to initiate new rounds
`of infection. Chlamydial physiology, struc-
`ture, developmental biology, and genetics are
`poorly understood. The limited and obligate
`intracellular growth of chlamydiae and the
`lack of any direct or indirect genetic methods
`for their study has restricted the development
`of biological and molecular understanding of
`these unusual organisms (4).
`The sequenced chlamydial genome consists
`of a 1,042,519–base pair chromosome (58.7%
`A⫹T) and a 7493–base pair plasmid (sequence
`and annotation available at http://chlamydia-
`www.berkeley.edu:4231 and GenBank under
`accession number AE001273). Analysis of the
`chlamydial genome resulted in the identifica-
`tion of 894 likely protein-coding genes (5).
`Similarity searching permitted the inferred
`functional assignment of 604 (68%) encoded
`proteins, and 35 (4%) were similar to hypothet-
`ical proteins deposited for other bacteria. The
`remaining 255 (28%) predicted proteins were
`not similar to other sequences deposited in Gen-
`Bank. Clustering by sequence
`similarity
`showed that 256 chlamydial proteins (29%)
`belong to 58 families of similar genes within
`the genome (paralogs), a fraction similar to
`other bacteria with relatively small genomes
`such as the mycoplasmas and Haemophilus
`influenzae (6). A list of the results of analysis of
`the predicted chlamydial proteins classified in
`accord with the functional systems in this bac-
`terium and a linear map of genes are available
`(5). The most prominent findings are presented
`below.
`Counterparts of enzymes characterized in
`other bacteria (orthologs) were identified in C.
`trachomatis to account for the minimal require-
`ments for DNA replication, repair, transcrip-
`tion, and translation. DNA repair and recombi-
`nation systems were extensively represented in
`the chlamydial genome, indicating that chla-
`mydiae have considerable recombination capa-
`bilities. There are also two predicted DNA he-
`
`754
`
`23 OCTOBER 1998 VOL 282 SCIENCE www.sciencemag.org
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`An MTP Inhibitor That Normalizes Atherogenic Lipoprotein Levels in
`WHHL Rabbits
` et al.John R. Wetterau
`
`282
`Science
`, 751 (1998);
`DOI: 10.1126/science.282.5389.751
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