Professional Documents
Culture Documents
DISCOVERY
STRATEGIES
METHODS
EDITED BY
ALEXANDROSMAKRIYANNIS
DIANEB~EGEL
Center for Drug Discovery
University of Connecticut
Storrs, Connecticut, U.S.A.
ISBN: 0-8247-0691-9
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Alexandros Makriyannis
Diane Biegel
Preface
Contributors
Xiayang Qiu
SmithKline Beecham Pharmaceuticals, King of Prussia,
Pennsylvania, U.S.A.
Sherin S. Abdel-Meguid
Suntory Pharmaceutical Research Laboratories, Cambridge,
Massachusetts, U.S.A.
I. INTRODUCTION
A. Crystallization
Obtaining large single crystals that diffract to high resolution remains the
primary bottleneck of protein crystallography. The most widely used
amino acid side chains can be readily assigned even in the absence of
sequence information.
Armed with the crystal structure of the protein–ligand complex and up-
to-date computer modeling software, one can design additional ligands.
Numerous molecular modeling software programs are available for that
purpose. However, it is important to note that current computational
algorithms have their limitations and utilize many approximations. There-
fore, while computer modeling software has been proven useful [4,18],
further testing and structural validations are required to identify the best
possible compound.
Any summary of experience gained during the last 15 years in the area of
structure-based drug design is in some way a work in progress, and clearly
there is much that we still need to learn.
VIII. OUTLOOK
Structure-based drug design is now an integral part of most if not all drug
discovery programs. It is a given that structure-based design is part of each
drug discovery effort whenever the target is a soluble protein. However, a
large segment of targets—namely, membrane proteins, particularly G-
protein-coupled receptors—are excluded. It is hoped that this situation will
be remedied in the near future.
REFERENCES
Angiotensin (AT1, AT2) Receptor tyrosine kinases NF-nB, STAT, NFAT, SMAD, CREB
Bradykinin (B1, B2) Epidermal growth factor
Cholecystokinin (CCKA) Fibroblast growth factor Proteases
Gastrin (CCKB) Insulin Aspartic proteases
Endothelin (ETA, ETB) Nerve growth factor Pepsin
a–Melanotropin (MCR1) Platelet-derived growth factor Renin
Adrenocorticotropin (MCR2) Cathepsins (D, E)
Substance P (NK1) Nonreceptor tyrosine kinases HIV-1 protease
Neurokinin-A (NK2) Src and Src family (Lck, Hck)
Neurokinin-B (NK3) Abl, Syk, Zap-70 Serinyl proteases
y-opioid (Enkephalin) Trypsin
A-opioid (Endorphin) Receptor serine/threonine kinases Thrombin
n-opioid (Dynorphin) Transforming growth factor Chymotrypsin-A
Oxytocin Kallikrein
Somatostatin (sst1–sst5) Nonreceptor serine/threonine kinases Elastase
Vasopressin (V1A, V1B) cAMP-Dependent protein kinase Tissue plasminogen activator
Neuropeptide-Y (Y1-Y5) Phosphoinositol-3-kinase (P13K) Factor Xa
Calcitonin Cyclin-dependent kinases (CDKs)
Molecular insight into the protein conformation states of Src kinase has
been revealed in a series of x-ray crystal structures of the Src SH3–SH2–
kinase domain that depict Src in its inactive conformation [7]. This form
maintains a ‘‘closed’’ structure, in which the tyrosine-phosphorylated
(Tyr527) C-terminal tail is bound to the SH2 domain (Fig. 2). The x-ray
data also reveal binding of the SH3 domain to the SH2–kinase linker
[adopts a polyproline type II (PP II) helical conformation], providing
additional intramolecular interactions to stabilize the inactive conforma-
tion. Collectively, these interactions cause structural changes within the
catalytic domain of the protein to compromise access of substrates to the
catalytic site and its associated activity. Significantly, these x-ray structures
provided the first direct evidence that the SH2 domain plays a key role in
the self-regulation of Src.
The bone disease osteoporosis results when an imbalance occurs in
the normal course of bone remodeling, a dynamic and highly regulated
process that involves both bone degradation (resorption) and bone for-
mation. Aberrantly high levels of bone resorption are associated with this
disease, which effects a net decrease in bone density and volume, resulting
in fragile, brittle bones that are subject to premature breaks and fractures
[8]. The most compelling evidence that Src is intimately involved in bone
remodeling comes from genetically engineered Src knockout mice. In these
Src (–/–) mice, the only major phenotype observed is excessive bone
-Asp-Gly-[pTyr-Aaa-Bbb-Ccc]-Ser-Pro-
(pY)(pY+1)(pY+2)(pY+3)
to advance Src SH2 inhibitors (see later: Sec. IV, Lead Discovery and
Combinatorial Chemistry).
The only direct ligand–protein hydrogen bond contact involves the
backbone NH of the pY+1 Glu with the carbonyl oxygen of the His 204
residue. In addition to the hydrophobic interactions involving the Ile
phosphopeptide residue and the pY+3 pocket, there exist potential
hydrogen-bonding possibilities from Tyr205, Ile217, and a buried
Tyr233 residue. Finally, two structural water molecules provide hydro-
gen-bonding networks between the pY+1 Glu (CO) and pY+3 Ile (NH)
phosphopeptide residues, and the Lys206 (NH) and Ile217 (CO) Src SH2
protein residues, respectively. Such structural waters act as drug design
elements to increase binding affinity (through favorable entropic contri-
butions) and can be exploited by small molecules that bind to or displace
them (see later: Sec. VI, Structure-Based, Small-Molecule Libraries to
Explore Src SH2 Binding).
The importance of the pTyr group for SH2 binding is counter-
balanced by the biological instability of the phosphate group to cellular
compounds are then synthesized and tested in the appropriate assays. The
biological data are analyzed in the context of available (x-ray or NMR)
structural information to impact the design of the next series of ana-
logues. This process is repeated until a lead compound or series of com-
pounds possessing the desired biological activities are obtained.
The database of available structural information during ARIAD’s
initial investigation into compounds targeting Src SH2 was limited; cases
involving ligand complexes utilized only peptide molecules [21]. Motivated
by an interest to develop orally active Src inhibitors (i.e., nonpeptides) we
adopted an exploratory approach to small-molecule lead discovery, using a
combinatorial chemistry strategy. Combinatorial libraries were biased
with a common phenyl phosphate group and systematically engineered
with diversity elements (selection guided by modeling) to probe the protein
surface for existing and new binding interactions (Fig. 7). Solid phase array
synthesis encompassing a novel phosphate ester linker strategy [22] was
Scheme 1
2a p-(FmocNHCH2CH2)Ph 0.659 93
2b p-(FmocNHCH2)Ph 0.637 89
2c m-(FmocNHCH2)Ph 0.627 88
2d p-(Allyl-O2CCH2CH2)Ph 0.730 92
2e p-(Allyl-O2CCH2)Ph 0.672 84
3f m-(Allyl-O2CCH2)Ph 0.807 98
3g p-[ p-(FmocNHCH2)PhO]Ph 0.552 81
3h p-(Allyl-O2CCH=CH)Ph 0.535 66
3i m-(Allyl-O2CCH=CH)Ph 0.796 98
Source: Ref. 22.
Figure 11 List of some of the molecular diversity building blocks used in the
construction of the nonpeptide phenyl phosphate libraries.
contacts with Lys182 (206 in Src) and Ile193 (217 in Src). The phenyl ring
of the benzamide template also forms favorable stacking interactions with
Tyr181 (205 in Src). Although the cyclohexylmethyl group interacts with
the pY+3 pocket, the contacts are primarily surface type and do not
extend as deeply into the pocket as the Ile of pTyr-Glu-Glu-Ile. Con-
sequently, SAR exploration of the pY+3 pocket, which had not been
rigorously studied with nonpeptide (peptidomimetic) small molecules
[13,14], became the first objective to be investigated.
Parallel synthesis provides the means of rapidly preparing discrete
analogues for both lead generation and lead optimization strategies,
which makes it an attractive option for developing compound databases
for therapeutic targets. Furthermore, the incorporation of structure-based
methods into the design and evaluation of parallel synthetic libraries has
proven to be a successful strategy for integrating the two drug discovery
technologies [29]. For the synthesis of the benzamide-containing com-
pounds, we devised a hitherto unreported solid phase, parallel synthetic
The next logical step in the progression to a cellularly active Src SH2
inhibitor was to incorporate a high affinity, biologically stable pTyr
mimic into the benzamide template. Drug design efforts at ARIAD led to
a novel Src SH2 inhibitors containing 4-diphosphonomethylphenylala-
nine (Dmp), namely, compound 25 (AP21773; Fig. 17) [16]. The design
concept for the Dmp group evolved from a 1.5 Å x-ray structure of Src
SH2, crystallized from citrate buffer, that fortuitously contained a citrate
molecule bound in the pTyr pocket. The x-ray structure reveals a number
of additional hydrogen bonds that citrate makes compared with a pTyr
group; this inspired the design of the Dmp moiety as a novel mimic of the
citrate interactions. Armed with these designed hydrogen bond contact
groups, we expected the Dmp to bind with greater affinity than pTyr, and
the Src SH2 binding results for AP21773 (Dmp) and AP21733 (pTyr)
confirm this prediction (Figs. 13 and 17). X-ray and NMR structural
studies involving AP21773 [16] verify these additional Dmp-related
contacts in the pTyr pcket, as well as other key Src SH2 interactions
observed earlier with this benzamide class as already discussed. The Dmp
moiety not only increases Src SH2 binding affinity, but also provides a
mechanism for tissue selectivity by targeting bone [16,35]. This targeting
feature provides a higher local concentration of compound on bone than
Figure 18 Solid phase synthetic scheme and molecular diversity groups for
compound 27. (From Ref. 36.)
ACKNOWLEDGMENTS
REFERENCES
Paul R. Gooley
University of Melbourne, Parkville, Victoria, Australia
I. INTRODUCTION
The assignment of the 1H, 13C, and 15N resonances depends on acquiring
a large number of separate 3-D or 4-D triple resonance experiments. The
experiments can be divided into intraresidue and interresidue and, when
combined, lead to sequence-specific assignment through bonds (Fig. 3) [1].
Improvements and new pulse sequences continue; however, a common set
of experiments to assign the backbone (frequently defined as Ha, Ca, N,
HN, C’, Ch) resonances of a protein are: 3-D HNCACB, CBCA(CO)NH,
HCACO, HNCO, (HCA)CO(CA)NH, 4-D HCANNH and HCA(CO)
NNH [29 –32]. Side-chain resonances are assigned using HCCH-TOCSY
and HCCH-COSY [33]. Unambiguous stereospecific assignment of the
methyl groups of Leu and Val are possible by preparing a 10% 13C-labeled
sample and acquiring a 1H,13C-HSQC spectrum [34]. The incorporation
of label is nonrandom such that Leu and Val residues are labeled as
13
Cy2H3-12CgH, 13Cy1H3-13CgH, and 13Cg2H3-12ChH, 13Cg1H3-13ChH,
respectively. Consequently, the 13Cy2H3 of Leu and 13Cg2H3 of Val
groups appear as singlets in the 1H,13C HSQC spectra and are thus readi-
ly stereoassigned. Measurement of 3JHNa in 3-D HNHA spectra [35] aids
determination of f torsion angles and stereoassignment of h-methylene
groups requires 3-D HNHB [36] and HACAHB [37] experiments. To
determine the fold of the protein, a large number of interresidue NOEs
must be assigned in 3-D 15N-NOESY [38], 3-D and 4-D 13C-NOESY ex-
periments [39,40]. The assignment of the backbone resonances of the
13 15
C, N-enriched catalytic domain of stromelysin-1 were mostly accom-
plished with 4-D HCANNH and HCA(CO)NNH experiments (Fig.4) [5].
Side-chain atoms were assigned with 3-D HCCH-COSY and HCCH-
TOCSY experiments with the carrier located near 35 ppm for aliphatic
side chains and at 124 ppm for aromatic side chains. Stereospecific
assignment of the methyl groups were obtained with a 10% 13C-labeled
Peak intensity data from NOE experiments were accumulated and con-
verted to interproton distances by calibrating against the expected short
distances in secondary structure elements. These data were complimented
with coupling constants determined from the 3-D HNHA and HNHB
experiments. A total of 2589 peaks were assigned in all NOE experiments.
After removal of nonconstraining and ambiguous NOEs, typically found
in mobile regions, 1814 meaningful restraints remained: 325 intraresidue,
429 sequential, 324 short-range (i+2 to i+5), 665 long range ( > i+5), and
71 intermolecular. Using a gridsearch program [44] 379 dihedrals (140 f,
140 c, 99 m1) were generated from sequential and intraresidue NOEs and
coupling constant data from HNHA and HNHB experiments. Structures
were calculated using the variable target function algorithm DIANA [45],
but it should be noted that in recent years this method has been replaced by
torsion angle dynamics methods that are far more efficient [46,47]. To
determine the structure of the complex, a residue template of the inhibitor
was built as a single residue covalently linked through an oxygen of the
carboxylate moeity of the inhibitor (Fig. 1) to the zinc which was
covalently bonded to the Nq2 of His-201. The residue template of His-
151 was created with the structural zinc covalently attached to its Nq2
atom. The structure calculation process is largely iterative with trial struc-
tures calculated and incompatible NOEs reassigned or removed and new
NOEs assigned on the basis of agreement with the trial structure. In the
final calculations, and to reduce bias in structure selection, plots of rmsd
and number of structures versus target function [48] of the final 80
pulse. The phase cycling of this pulse with respect to the receiver determines
whether X-nucleus attached protons are selected or filtered. If both signals are
added to the receiver (x,x) X-nucleus attached protons are filtered; and if the
receiver phase is alternated (x,-x) the X-nucleus attached protons are selected. (B)
A doubly tuned half filter for filtering 13C attached protons [42]. In this ex-
periment the filter consists of two delays (H 1,H 2) tuned to different 1JCH values
resulting in superior suppression of artifacts. (C) A doubly tuned time-shared half
filter for 13C/15N (43). In this experiment D = 1/(41JNH), D1 = 1/(41JCH) and D2 =
[1/(41JNH 1/(41JCH)]. Phase cycling the receiver selects or filters both 13C and
15
N attached protons.
one antiparallel strand and the topology 1x, +2x, +2, 1, using
the Richardson nomenclature [50]. The h-sheet lies on two helices (helix
A and B); a third helix (helix C) is near the C-terminus. The molecule
has two zincs: a catalytic zinc is located at the bottom of a cleft, and
a structural zinc above the h-sheet. The overall fold of sfSTR may
be described as follows. The N-terminus is located near the N-terminal
end of helix C. The protein backbone forms a poorly defined irregular
strand for the first 13 residues before entering strand I of the h-sheet,
then descending through helix A. Helix A acts as a backbone to the
protein, spanning its full length. The pronounced amphipaticity of this
helix provides hydrophobic residues for internal packing to helix B and
to the h-sheet, and the hydrophilic residues are exposed to the solvent.
After helix A the protein backbone turns to form strand II of the h-sheet,
which lies parallel to and outside strand I. This strand rises steeply, giving
the h-sheet a distinctly twisted appearance. It is connected by a short
loop to strand III, which is parallel to and inside of strand I. A long loop
connects strands III and IV, crossing over strand V and placing strand IV
along the ligand-binding cleft and antiparallel to strand V. Another small
ligated by His-166 from strand IV, His-179 from strand V, and His-151 and
Asp-153 both from a 14 residue loop. The catalytic zinc is ligated by His-201
and – 205 from helix B and His-211. (B) The complex is viewed from below S1V
subsite. The heavy atoms of the inhibitor and the residues that are in
intermolecular contact (Leu-164, Leu-197, Val-198, His-201, Leu-218, Tyr-220,
Leu-222, Tyr-223) with the P1V homophenylalanine are shown. To reduce
crowding in the figure not all these residues are labelled. (*) marks Leu-218
and His-201. Val-198 is below Leu-197. Leu-164 is at the N-terminal end of
the h-strand that appears above Leu-197 in this figure. The ribbon diagrams
were produced by MOLSCRIPT [62].
the angle to 35j. The analysis suggests that the NH of the P3V hydrogen
bonds to the carbonyl of Asn-162; the carbonyl of P1V hydrogen bonds to
the NH of the Leu-164 (which is slowly exchanging with deuterium); and
the amine of P1V hydrogen bonds with the carbonyl of Ala-165. The
structures described here do not show hydrogen bonds between the NH
and the carbonyl of the P2V arginine to the protein, which is in contrast to
reported crystal structures which show a hydrogen bond to the NH of Tyr-
223 [10]. Although Pro-221 and Tyr-223 are near atoms of the inhibitor, for
example, the NH of Tyr-223 shows weak NOEs to the ring of P3V, their
distances in the structure models are not in agreement with these residues
participating in hydrogen bonds. The NH of Tyr-223 does not show slow
exchange with 2H2O and analysis of 2-D saturation transfer difference
1
H,15N HSQC spectra suggested that the exchange rate of the NH of Tyr-
223 was one to two orders slower than a free amide proton further
VIII. CONCLUSION
This chapter has discussed the use of heteronuclear NMR and isotope
editing methods to determine the structure of protein complexes of
therapeutically important drug targets. NMR methodology continues
to develop with larger protein complexes being studied, and more
accurate structures being determined. Developments include deuteration
of proteins [1] to enhance relaxation properties, and experiment design,
for example, Transverse Relaxation Optimized Spectroscopy (TROSY)
[55], which takes advantage of favorable relaxation pathways thus
allowing proteins of at least 60 kDa to be studied; inclusion of residual
dipolar couplings as an orientation constraint in structure calculations
[56,57] are increasing the accuracy of solution structures; and combining
deuteration and TROSY experiments has allowed hydrogen bonds to be
directly observed and also included in structure calculations [58]. An
REFERENCES
Alexandros Makriyannis
University of Connecticut, Storrs, Connecticut, U.S.A.
Andreas Goutopoulos
Serono Reproductive Biology Institute, Rockland, Massachusetts, U.S.A.
I. INTRODUCTION
Cannabis sativa, one of the oldest plants farmed by man, has been known
for its medicinal properties for at least four millennia (Peters, 1999). The
psychoactive–euphoric effects of this plant, as well as its facile and wide
climatic range of cultivation, have rendered it a very popular recreational
drug. Today, cannabis, or marijuana, is still the focus of strong social,
legal, and medical controversy over its therapeutic utility.
Referenda in Arizona and California in 1997, and later, others in
eight additional states, aimed at legalizing marijuana cigarettes for medical
purposes. Two licensed, single-compound, cannabimimetic pharmaceuti-
cals, Marinol (Dronabinol, delta-9-THC from Roxane Labs) and Cesamet
(Nabilone, developed at Eli Lilly, now in use in the United Kingdom), are
marketed for two purposes: to control the nausea and emesis produced by
cancer chemotherapy and to serve as appetite stimulants in AIDS-related
anorexia. In clinical trials with cancer chemotherapy patients, both these
agents have proven to be superior to conventional antiemetics, such as
perchlorperazine (Breivogel, 1998).
Beyond this relatively limited medical use of cannabimimetics, the
current, albeit long-delayed elucidation of their pharmacology is likely to
lead to a wide expansion of the clinical potential and significance of
(Schmid, 1997). Very low levels have been detected in serum, plasma, and
cerebrospinal fluid—a fact that indicates that anandamide is not hormonal
in nature but rather is biosynthesized at or near its sites of action.
In addition to anandamide, several other endogenous polyunsatu-
rated fatty acid derivatives were also found to act as cannabimimetics.
They are all now collectively referred to as endocannabinoids. Soon after
the discovery of anandamide, two more fatty acid ethanolamides were
isolated and found to bind to CB1 preparations with affinities similar to
that of anandamide (anandamide CB1 binding affinity Ki = 39.2 nM,
according to Hanus et al., 1993). These were the homo-g-linolenylethanol-
amide (CB1 Ki = 53.4 nM) and 7,10,13,16-docosatetraenylethanolamide
(CB1 Ki = 34.4 nM) (Fig. 2). All three N-acylethanolamide endocanna-
binoids were found to be CB1 agonists in the MVD test (Pertwee, 1994).
A different type of endocannabinoid that is also an arachidonic acid
derivative was first isolated from canine gut and identified as 2-arachidonyl
glycerol (2-AG) (Fig. 3) (Mechoulam, 1995a).
Later 2-AG was also found in the brain (Stella, 1997) and spleen
(Di Marzo, 1998). It was shown to be released in a calcium-dependent
manner, reaching concentrations 170 times higher than that of ananda-
mide in the brain (Stella, 1997). Like the other endocannabinoids, 2-AG
was shown to produce the typical tetrad of cannabimimetic behavioral
effects and inhibit electrically evoked contractions of mouse MVD
(Mechoulam, 1995a).
A. Anandamide Pharmacology
Since the discovery of anandamide in 1992, a number of studies have
examined its pharmacological properties. Although its roles are still
elusive, a plethora of data supports the initial postulate that anandamide
is the major endogenous cannabinoid ligand. As mentioned earlier, anan-
damide binds to CB1 from brain preparations and displaces various well-
B. Endocannabinoid Metabolism
Biosynthesis of Anandamide
Considerable advances have been made during the late 1990s toward
understanding the physiological pathways that are involved in the syn-
thesis and inactivation of endocannabinoids. The first of these pathways to
be observed, an enzymatic activity responsible for anandamide hydrolysis,
led to lower apparent CB1 affinities for anandamide analogs in studies
involving structure–activity relationships (SARs) (Childers, 1994),
(Abadji, 1994). Inclusion in the binding assay of phenylmethanesulfonyl
fluoride (PMSF), a general serine protease inhibitor, protected the anan-
damide analog from hydrolysis (Abadji, 1994; Khanolkar, 1996). Shortly
after, an enzyme specific for this hydrolytic process was identified and
characterized (Deutsch, 1993; Ueda, 1995). Initially, it was thought that
this hydrolase, named anandamide amidase or fatty acid amidohydrolase
(FAAH), was also responsible for the synthesis of anandamide by acting
reversibly (Devane, 1994). However, the current belief is that anandamide
amidase is unlikely to be physiologically responsible for anandamide
synthesis because of the requirement for significantly higher than normal
Biosynthesis of 2-AG
Two possible pathways for the biosynthesis of 2-AG have been proposed:
(1) a phospholipase C (PLC) hydrolysis of membrane phospholipids
followed by a second hydrolysis of the resulting 1,2-diacylglycerol by
diacylglycerol lipase or (2) a phospholipase A1 (PLA1) activity that
generates a lysophospholipid, which in turn is hydrolyzed to 2-AG by
lysophospholipase C (Fig. 5) (Piomelli, 1998). Alternative pathways may
also exist from either triacylglycerols by a neutral lipase activity or
lysophosphatidic acid by a dephosphorylase. The fact that PLC and
diacylglycerol lipase inhibitors inhibit 2-AG formation in cortical neurons
supports the contention that 2-AG is, at least predominantly, biosynthe-
sized by the PLC pathway (Stella, 1997). However, a mixed pathway may
also be plausible.
As with the biosynthesis of anandamide, the biosynthesis of 2-AG is
also triggered by increases of intracellular calcium ions that result from
neuronal activity. High frequency stimulation of neurons produced a
fourfold increase of 2-AG accumulation compared with controls, and this
was prevented by sodium ion channel blocking or removal of calcium ions
(Stella, 1997). The concentration of 2-AG in depolarized neurons reached 1
to 2 AM, significantly higher than anandamide and sufficient to substan-
tially activate CB1 (Stella, 1997).
Based on the pathways just proposed for the biosynthesis of anan-
damide and 2-AG, the formation of these endogenous ligands must also be
dependent on the composition of the precursor lipids. This dependence is
of greater importance for anandamide rather than for 2-AG because
Endocannabinoid Release
Immediately after synthesis, endocannabinoids are released in the extra-
cellular space, where they then act on the same or neighboring cells as
autocrine or paracrine mediators (Di Marzo, 1999). Experimental evidence
thus far indicates that anandamide and 2-AG, unlike other classical
neurotransmitters, are not stored in vesicles. First, anandamide basal
concentrations are extremely low (5–10 pmol/g), 100 to 10,000 times lower
than those of classical neurotransmitters (Cadas, 1997). Second, stimu-
lus-dependent anandamide release is linked with de novo NAPE and
Endocannabinoid Inactivation
Anandamide is inactivated in two steps, first by transport inside the cell
and subsequently by intracellular enzymatic hydrolysis. The transport of
anandamide inside the cell is a carrier-mediated activity, having been
shown to be a saturable, time- and temperature-dependent process that
involves some protein with high affinity and specificity for anandamide
(Beltramo, 1997). This transport process, unlike that of classical neuro-
transmitters, is Na+-independent and driven only by the concentration
gradient of anandamide (Piomelli, 1998). Although the anandamide trans-
porter protein has not been cloned yet, its well characterized activity is
known to be inhibited by specific transporter inhibitors. Reuptake of 2-AG
is probably mediated by the same facilitating mechanism (Di Marzo,
1999a,b; Piomelli, 1999).
Once inside the cell, anandamide is hydrolyzed by a specific hydro-
lase, anandamide amidase (AEAase) or fatty acid amidohydrolase
(FAAH) (Desarnaud, 1995; Deutsch, 1993). This enzyme is membrane
associated and shows significant specificity for anandamide (Desarnaud,
1995; Lang, 1999).
There is some evidence that in cells with low anandamide amidase
activity, such as platelets and neutrophils, anandamide is inactivated by an
oxidative pathway involving 12(S)-lipoxygenase (Edgemond, 1998).
Metabolism of anandamide by enzymes of the arachidonic acid cascade
A. Classical Cannabinoids
Classical cannabinoids (CCs) are tricyclic terpenoid derivatives bearing
a benzopyran moiety. This class includes the natural product (–)-delta-
nine-tetrahydrocannabinol (Fig. 8, 1) and the other pharmacologically
active constituents of the plant Cannabis sativa.
B. Nonclassical Cannabinoids
A second class of cannabimimetics was developed at Pfizer, in an effort to
simplify the structure of CCs while maintaining or improving activity
(Johnson, 1986). This class includes bicyclic (e.g., 6) and tricyclic (e.g., 7)
analogs lacking the pyran ring of CCs (Fig. 9). These compounds are
collectively specified as ‘‘non-classical cannabinoids’’ (NCCs). The crys-
talline CP55,940 (6) and its tritiated analog show high affinity, efficacy, and
stereoselectivity to both cannabinoid receptors and have been used exten-
sively as pharmacological tools. The key compound that led to the
discovery of CB1 was [3H]CP55,940 (Devane, 1988).
The structural resemblance of NCCs and CCs, as well as their
comparable SARs, indicate that they bind to CB1 in a similar fashion.
The side chain and the phenolic hydroxyl of an NCC are crucial for
activity. The hydroxypropyl chain of CP55,940 is not necessary for
C. Aminoalkylindoles
The third chemical class of cannabinergics is that of aminoalkylindoles
(AAIs) (Fig. 10). They were developed at Sterling Winthrop as potential
nonsteroidal anti-inflammatory agents (Bell, 1991). These first analogs
exhibited antinociceptive properties that were eventually attributed to
interactions with the cannabinoid receptors. Compound 8 (WIN55212)
is a potent CB1 and CB2 agonist with high stereoselectivity and a slight
preference for CB2. AM630 (9), the first CB2-selective antagonist derived
from this class of compounds, was developed in our laboratory after long-
term efforts to obtain such an inhibitor (Pertwee, 1995a). We have recently
reported the development of AM1241, a potent, highly CB2-selective
agonist (Malan, 2001).
This class of compounds differs from the first two by being consid-
erably less lipophilic and more ‘‘druglike.’’ Labeling of CB1 with electro-
philic AAIs almost abolished the receptor’s ability to bind to CP55,940,
indicating that AAIs and NCCs (as well as CCs) share at least some points
of interactions with CB1 (Yamada, 1996). Several models have attempted
to define the pharmacophoric equivalency between the functional groups
of AAIs, NCCs, and CCs (Xie, 1995), (Huffman, 1994). Although these
three different classes of cannabimimetics show similarities in their
binding with CB1, they differ considerably in the susceptibility of their
binding affinities to different Na+-modulated allosteric receptor states
(Houston, 1998). They also differ in their affinities to several CB1
mutants (Chin, 1998), as well as in the way they activate the receptor
(Houston, 1998). These differences may be explained by the existence of
more than one ligand binding motif, or by ligand binding to partially
overlapping but distinct receptor binding subsites, or even by induction
of different receptor conformational changes upon binding of different
ligands (Howlett, 1998a). It has been proposed that structurally dissim-
ilar ligands may evoke different receptor–G-protein coupling (Houston,
1998). Therefore, analogs from different cannabinoid ligand classes may
evolve as selective pharmacological agents exhibiting only specific can-
nabimimetic effects.
Structural features of AAI important for cannabinergic activity are
the 3-aroyl moiety and the 1-chain, which must contain nitrogen, most
often in a heterocyclic ring (e.g., piperidino or morpholino). This chain can
D. Endocannabinoids
The class of the endogenous cannabinoids (endocannabinoids) was dis-
covered in 1992 as molecules produced by mammalian cells with affinity for
the cannabinoid receptor (Devane, 1992). This class includes lipid mole-
cules such as fatty acid ethanolamides, monoacylglycerols, and related
synthetic analogs. The two prototypes in this class are the ethanolamide of
arachidonic acid (anandamide) and 2-arachidonyl glycerol (2-AG). Its (R)-
1V-methylated analog, AM356 (10) (Fig. 11) shows higher affinity and
remarkable metabolic stability (Abadji, 1994). This analog, named R-
methanandamide, has been established as a standard CB1-selective agonist
in the cannabinoid field. The (R,R)-2,1V-dimethyl anandamide was
reported recently to exhibit a threefold improved affinity over R-meth-
anandamide and significant enantioselectivity (Goutopoulos, 2001). Other
modifications that result in high CB1 affinity include the substitution of the
hydroxyl group with halogen, or the methyl group, and the substitution of
the terminal n-pentyl chain with the dimethylheptyl chain, reminiscent of
potent classical cannabinoid ligands (e.g., 12, O-1064) (Pertwee, 2000).
This compound class also includes some fatty acid analogs designed for
endocannabinoid targets other than the cannabinoid receptors. For
instance, arachidonyltrifluoromethylketone (ATFMK) (13) and hexade-
cylsulfonyl fluoride (14, AM374) are potent inhibitors of anandamide
amidase. The first inhibitor of the anandamide transporter to play an
important role in the discovery of this transport process was AM404 (15)
(Beltramo, 1997).
E. 1,5-Biarylpyrazoles
The fifth class, 1,5 biarylpyrazoles, was developed at Sanofi in 1994 from a
hit generated by high throughput screening for cannabinoid receptor
ligands (Rinaldi-Carmona, 1994). Compounds of this class act as canna-
binoid receptor antagonists. Figure 12 shows SR141716A (16), which was
reported, simultaneously with AM630, as the first CB1 antagonist and has
since been used extensively as an important pharmacological tool.
SR141716A shows selectivity for CB1 and often acts as an inverse agonist
rather than a pure antagonist (Pertwee, 2000). Also developed at Sanofi,
SR144528 (17) acts as an antagonist/inverse agonist with selectivity for
CB2. A useful radioimaging agent in PET and SPECT studies [123I]
A. Nervous System
The primary system of cannabimimetic activity is the nervous system. The
CB1 receptor is omnipresent in the brain, especially in areas that control
functions affected by cannabimimetics. One of the functions most pro-
nouncedly influenced by cannabimimetics is motor behavior. Catalepsy,
immobility, ataxia, and impairment of complex behavioral acts after acute
administration of high doses of cannabimimetics are manifestations of
such motor effects (Pertwee, 1997). In lower doses cannabimimetics
produce the opposite effects. The very dense presence of CB1 in the
cerebellum and the basal ganglia, areas responsible for motor activity, is
B. Immune System
The discovery of the peripheral CB2 receptor, which localizes in cells of the
immune system, is very likely linked to the well-known immunosuppres-
sion of marijuana smokers.
Miskin (1985) found that delta 9-THC decreases host resistance to
herpes virus type 2 in mice and guinea pigs by decreasing both cellular and
humoral immunity. In vivo and in vitro studies indicate that macrophages
are the major targets of cannabinoids. delta 9-THC inhibits, in a dose-
dependent manner, the extrinsic antiviral activity of macrophages (Cabral,
1991). It was also shown that cannabinoids cause morphological changes
in macrophages (Cabral, 1991) and affect their phagocytic and spreading
ability (Spector, 1991).
The involvement of CB2 (and possibly of CB1) receptor(s) in the
immunosuppressive effects of cannabinoids is not proven yet. The local-
ization of CB2 in cells of the immune system and especially in macrophages
and lymphocytes suggests that this receptor serves some immunoregula-
tory role(s). The first strong piece of evidence that implicates CB2 in such a
function came from Kaminski et al. (1994), who demonstrated that
cannabinoid-induced suppression of humoral immunity was partially
mediated through inhibition of adenylyl cyclase by a G-protein-coupled
mechanism that is pertussis toxin sensitive. Involvement of a membrane
perturbation mechanism in cannabinoid-induced immunosuppression is
also possible, especially in areas exposed to high drug concentrations, such
as lung alveolar macrophages of marijuana smokers (Cabral, 1999). The
involvement of the cannabinoid system in the regulation of the immune
system may suggest that cannabinergics could potentially serve as immu-
nomodulatory agents. Although CB2 selective agents already exist, their
clinical potential in some immunomodulatory role will not be realized until
the CB2 physiological functions are better understood. Cannabidiol, a
cannabis terpenoid ingredient lacking the pyran ring as well as significant
binding affinity for CB1 and CB2, was shown to be an active anti-
inflammatory agent in the murine model of arthritis (Pertwee, 2000). The
molecular basis of this observation is still unknown.
D. Reproductive System
Cannabinoids produce increased ring and chain chromosomal transloca-
tions and morphological abnormalities in mouse sperm, as well as reduc-
tion of sperm concentration in humans (Zimmerman, 1999). Strong
evidence indicates the presence of functional CB1, or CB1-like receptors,
in human sperm (Schuel, 1999). Furthermore, the endogenous cannabimi-
metic anandamide is produced in the human uterus and testes (Schuel,
1999). These findings along with several observations on cannabinoid-
induced effects on reproductive functions suggest that the cannabinoid
system may be directly involved in the regulation of sperm production,
sperm motility, the acrosome reaction, and prevention of polyspermy
(Schuel, 1999). The endocannabinoid system in the uterus appears to play
a fundamental role in embryo implantation and early development.
Anandamide inhibits these processes and, therefore, regulation of its
levels seems to control the timing of these events (Paria, 1995). These
findings are also in line with recent clinical observations that correlate the
levels of AEAase expression with miscarriages in pregnant women
(Maccarone, 2000). Further understanding of the endocannabinoid func-
tions in the reproductive system will open perspectives for exploitation of
cannabinergics for the treatment of some types of infertility or the devel-
opment of contraceptives.
Cannabimimetics are also shown to affect reproductive and
metabolic functions indirectly by hormonal modulation through the
hypothalamic and pituitary regulatory centers. They are found to
reduce serum levels of the luteinizing hormone, prolactin, growth
hormone, and thyroid-stimulating hormone, and to increase cortico-
tropin (Murphy, 1998).
F. Respiratory System
Cannabimimetics are known to produce bronchodilation, which is man-
ifested by a marked increase in airway conductance and reduction in
airway resistance (Vachon, 1973). Although the mechanism of this activity
is not known, it probably does not directly involve adrenergic receptors.
Possible involvement of CB1A (a CB1 variant found in the lung) in
cannabinoid-induced bronchodilation is still unexplored (Shire, 1995).
Recently, it was shown that anandamide is released in the lung upon
Ca2+ stimulation and exerts a dual effect on bronchial response. It strongly
inhibits capsaicin-evoked bronchospasm and cough; however, it causes
bronchoconstriction in vagotomized rodents (Calignano, 2000). These
effects are mediated by CB1 receptors present in axon terminals of airway
nerves since they are blocked by SR141716A. This endocannabinoid-
mediated control of airway responsiveness may be exploited in the devel-
opment of new antiasthmatic agents.
G. Gastrointestinal System
Cannabimimetics reduce the intestinal motility by a CB1-mediated inhib-
itory activity on acetylcholine release from autonomic fibers. An endo-
cannabinoid, 2-AG, was isolated from dog intestine; however, its role there
remains unknown (Mechoulam, 1995a).
VII. CONCLUSIONS
REFERENCES
1. Abadji V, Lin SY, Taha G, Griffin G, Stevenson LA, Pertwee RG, Makri-
yannis A. (R)-Methanandamide: a chiral novel anandamide possessing
higher potency and metabolic stability. J Med Chem 1994; 37:1889–1893.
2. Aceto MD, Scates SM, Razdan RK, Martin BR. Anandamide, an endog-
enous cannabinoid has a very low physical dependence potential. J Phar-
macol Exp Ther 1998; 287:598–605.
3. Arnone M, Maruani J, Chaperon F. Selective inhibition of sucrose nd
ethanol intake by SR141716a, an antagonist of central cannabinoid (CB1)
receptors. Psychopharmacology 1998; 132:104–106.
4. Baker D, Pryce G, Croxford JL. Cannabinoids control spasticity and
tremor in a multiple sclerosis model. Nature 2000; 404:84–87.
5. Baker D, Pryce G, Croxford JL, Brown P, Pertwee RG, Makriyannis A,
Khanolkar A, Layward L, Fezza F, Bisogno T, Di Marzo V. Endocanna-
binoids control spasticity in a multiple sclerosis model. FASEB J 2001; 15:
300–302.
6. Batkai S, Jarai Z, Wagner J, Goparaju S, Varga K, Liu J, Wang L, Mirshahi
F, Khanolkar A, Makriyannis A, Urbaschek R, Garcia N Jr, Sanyal A,
Kunos G. Endocannabinoids acting at vascular CB1 receptors mediate the
vasodilated state in advanced liver cirrhosis. Nat Med 2001; 7:827–833.
7. Bell MR, D’Ambra TE, Kumar V, Eissentat MA, Herrmann JL, Wetzel
JR, Rosi D, Philion RE, Daum SJ, Hlasta DJ, Kulling RK, Ackerman JH,
Haubrich DR, Luttinger DA, Baixman ER, Miller MS, Ward SJ. Anti-
nociceptive (aminoalkyl)indoles. J Med Chem 1991; 34:1099–1110.
I. INTRODUCTION
Relatively few human imaging studies have evaluated the effects of mari-
juana or THC on metabolism or blood flow. Acute intravenous THC in
both normal controls and habitual marijuana users led to increased an
increased regional cerebral metabolic rate (CMR) in the cerebellum. This
increase is positively correlated both with concentrations of THC in the
plasma and with the intensity of the subjective sense of intoxication [5].
In a 1997 PET/[15O]water study with 32 abusers [6], THC dose-depend-
ently increased cerebral blood flow (CBF) in the frontal regions, insula
The first attempt to develop a PET radioligand for imaging brain CB1
receptors involved modification of D8-THC by labeling with fluorine-18 in
the hydrocarbon side chain [16,17], as shown in Figure 2. Unfortunately,
Figure 7 Structure of an 18F-labeled pyrazole ligand; for brain uptake (see Table
1).
Figure 8 Comparison of ability of WIN 55, 212-2 to sedate mice (triangles) and
to block specific binding of [131I]AM281 (squares).
V. CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
I. INTRODUCTION
*Present address: AstraZeneca Research Centre Montreal, St. Laurent, Quebec, Canada.
Orn-Phe-Glu-NH2, was a full agonist at both the A and the y receptor but
showed a substantial decrease in potency. Finally, the conformationally
j j
j j
The first y antagonists derived from opioid peptides were obtained through
diallylation of the N-terminal amino group of enkephalin analogues. The
best known compound of this type is N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-
OH (ICI 174,864; Aib = a-aminoisobutyric acid), which is quite y selective
(K Ai /K yi = 128) but not very potent (K yi = 199 nM; Ke = 69 nM in the
MVD assay) [33,34]. The nonpeptide y antagonist naltrindole (NTI) [35] is
highly potent but displays only modest y selectivity (K Ai /K yi = 21.2). A
benzofuran analogue of naltrindole, NTB, showed improved y selectivity
but somewhat lower y-antagonist potency [36]. However, both NTI and
NTB also turned out to be antagonists against A and n agonists in the GPI
assay, with potencies (Ke = 29–48 nM) about 100 to 300 times lower than
those observed against y agonists in the MVD assay (Ke = 0.13 and 0.27
nM, respectively) [36]. The recently discovered TIP(P) peptides represent a
novel class of potent and highly selective y-opioid antagonists [37]. The two
prototype antagonists were the tetrapeptide H-Tyr-Tic-Phe-Phe-OH
(TIPP;Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and the
Ke K Ai K yi
Compound (nM)a (nM)b (nM)b K Ai /K yi
and shows even slightly higher y-receptor selectivity than the TIPP[C]
parent peptide.
For the purpose of opioid receptor binding studies, TIPP was also
radioiodinated. Surprisingly, [125I]TIPP binding to y receptors in N4TG1
neuroblastoma cells was substantially reduced in the presence of Na+ and
Gpp(NH)p [42]. These results indicated that substitution of an iodine atom
at the 3V position of Tyr1 in TIPP had turned the y antagonist into a y
agonist. The corresponding ‘‘cold’’ analogue, H-Tyr(3’-I)-Tic-Phe-Phe-
OH, was then synthesized and shown to be a full agonist in the MVD
assay (IC50 = 97 nM). This agonist effect was antagonized by TIPP (Ke =
11 nM) [38]. Corresponding iodination of the Tyr residue in TIP and
TIPP[C] did not result in agonism, but somewhat reduced antagonist
case [52]. The lowest energy conformers of both TIPP and TIPP[C]
showed good overlap of their Tyr1 and Tic2 aromatic rings and N-terminal
amino group with the corresponding pharmacophoric moieties of nal-
trindole. Thus, these results are in agreement with the model of the
receptor-bound conformation of TIP proposed earlier. This model is
characterized by all-trans peptide bonds and was definitely confirmed by
conformational analyses of two TIPP analogues (y antagonists) in which a
cis peptide bond between the Tyr1 and Tic2 residues is sterically forbidden
[53]. Both TIPP and TIPP[C] are very hydrophobic peptides, and the
results of the theoretical conformational analyses clearly indicated that
they enjoy considerable structural flexibility, particularly in their C-
terminal dipeptide segment. There is no doubt that their conformations
are quite dependent on the environment. According to our theoretical
analysis, a crystal structure of TIPP published in 1994 [54] is about 3 kcal/
mol higher in energy than the lowest energy structure and shows no
similarity to any of the calculated low energy structures [52,53]. The
crystal structure of TIPP appears to be stabilized by a large number of
intermolecular hydrophobic contacts between layers of TIPP molecules in
the crystal and by several hydrogen bonds to solvent (AcOH) molecules.
There is no reason to believe that it resembles the y-receptor-bound
conformation of TIPP. In an aqueous environment TIP(P) peptides
may undergo a so-called hydrophobic collapse [55]. It is possible that
subtle structural modifications, such as introduction of an iodine sub-
V. D AGONISTS
IC50 K yi K Ai
Compound (nM)a (nM)b (nM)b K Ai /K yi
VI. CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
Steven A. Kates
Surface Logix, Inc., Brighton, Massachusetts, U.S.A.
I. INTRODUCTION
Organic chemistry in the last half of the twentieth century has evolved to a
level of extreme sophistication in which complex macromolecules thought
only to exist in nature were prepared in a laboratory hood. The process
typically involves performing a reaction in an organic solvent followed by
isolating, purifying, and analyzing the compound. This tedious, time-
consuming procedure requires considerable expertise. Bruce Merrifield
was the first to recognize an alternative approach for the preparation of
organic compounds. He applied this method to synthetic peptides and was
awarded the Nobel Prize in 1984 for this discovery [1]. The concept was to
perform the chemistry proven in solution but add a covalent attachment
step that links the target to an insoluble polymeric support. Key advan-
tages to the solid-phase technique include simple filtration, washing with-
out manipulative losses, and ease of automation.
Peptide synthesis was amenable to solid-phase techniques since the
process was repetitive. The C-terminal amino acid is attached to polymeric
surface and the peptide chain is assembled via a two-step process: coupling
of the incoming amino acid that has the alpha-amino group protected
Figure 1
Figure 3
linker 10 (HMB) [14] contains a carbonyl group para to the ester anchor
and is activated to nucleophilic attack such as hydroxide ion and is stable
toward acid. Alkoxybenzyl derivatives with greater electron donor
strength (Fig. 6) such as SASRIN (super-acid-sensitive resin) 11 [15],
Rink acid 12 [16], and HAL (hyper-acid sensitive) 13 [17] resin allow
carboxylic acids to be cleaved using a lower acid concentration (typically
Figure 6
Scheme 1
for cleavage and carboxylic acids are released using TFA (Scheme 2). Resin
bound diazo linker 19 was synthesized starting from Wang resin and was
further oxidized to a benzyl aldehyde (Scheme 3) [23]. Carboxylic acids are
anchored to the support in a rapid, colorimetric reaction and are released
upon TFA treatment.
Photolabile linkers play an important role in solid-phase organic
synthesis (SPOS) due to their stability under both acidic and basic
conditions. The ONb photolabile linker was modified to improve cleav-
age rates and yields; Fmoc-Tos-OH was released in 87% yield after 23 h
(Scheme 4) [24]. Specifically, the primary alcohol was changed to a
secondary benzylic alcohol and the attachment to the resin was through
an alkyl chain as opposed to an amide function. Linker 20 was used for
the production of carboxylic acids or carbohydrates. A second example
Scheme 3
Scheme 5
Scheme 7
Scheme 9
Figure 9
Scheme 10
Scheme 12
Figure 11
Scheme 15
Scheme 17
Scheme 19
Scheme 21
Scheme 23
Scheme 24
Scheme 25
Scheme 26
Scheme 28
Scheme 29
Scheme 30
Scheme 31
Scheme 33
Scheme 34
The Leznoff acetal linker 69 was used to anchor an aldehyde to the solid
support and following a series of reactions, the aldehyde was released by
acidic cleavage [78]. An application of this resin was demonstrated for a
biaryl aldehyde library synthesis which incorporated a Suzuki–Miyaura
reaction (Scheme 37) [79]. Cleavage was effected by a solution of 3 M HCl
Scheme 36
Scheme 38
Scheme 40
Scheme 42
X. CONCLUSION
Angeliki P. Kourounakis
University of Thessaloniki, Thessaloniki, Greece
Pieter van der Klein and Ad P. IJzerman
Leiden University, Leiden, The Netherlands
I. INTRODUCTION
The term ‘‘allosteric’’ was first introduced by Monod and Jacob [6], who
referred to an allosteric inhibition (of the synthesis of a tryptophan
precursor by tryptophan) in describing the mechanism underlying the
action of ‘‘an inhibitor that was not a steric analog of the substrate.’’
Thus, first introduced in the field of enzymology, the term ‘‘allosteric’’
(Greek aEEo, other, different; jH eUeo, solid, shape) means ‘‘having a
different shape.’’ It soon referred to the presence in an enzyme of a
(secondary) site of attachment for a substance that modifies enzyme
activity without interacting directly with the active center (primary site).
The allosteric effect, therefore, was attributed to a change in either the
three-dimensional structure of the peptide chain or else a change in
affinity for the G protein, ligand A, and allosteric modulator (X). Relative
stoichiometry of the states would be determined by the presence of G
protein and agonists and modified by allosteric modulators [10].
low affinity of agonists [64–67]. This indicates that an intact agonist bind-
ing site of the receptor is required for PD81,723 to exert its allosteric
action [62].
Recently we developed a series of novel PD81,723 analogues,
some of which appear to be superior to PD81,723 in their enhancing
activity [68,69]. The synthesis of these derivatives is relatively straight-
forward, as shown in Figure 10 [68–72]. The 4,5-dimethyl group and
the benzoyl moiety were targets for further modifications, leading to
series of 4,5-dialkyl (1a–g), of tetrahydrobenzo (1h–u) and of tetrahy-
dropyridine (3a–g) derivatives (Fig. 10, Tables 1 and 2). These deriva-
tives were evaluated both as allosteric enhancers of agonist binding to
the rat adenosine A1 receptor and as antagonists on this receptor.
Among them, a number of compounds, in particular 1b, 2e, 1j, 1n,
and 1u (Fig. 11, Table 1), proved to be superior to the reference
compound (PD81,723) in both enhancing activity and diminished antag-
onistic behavior [68].
Some structure–activity relationships of a further developed R4, R5
alkyl/cycloalkyl series (2a–o, Fig. 10, Table 1) were also investigated.
This study [69] revealed structural features that favored allosteric
enhancing activity, such as benzoyl lipophilic substitution and thiophene
4-alkyl substitution, while other features, such as thiophene 5-bulky
substitution, favored antagonistic properties. Upon further analysis, a
a
Enhancing activity (at 10 AM of test compound) is expressed as percentage of decrease (FSEM) in
[3H]CCPA dissociation over control (0%) and that of PD81,723 (100%, n = 3).
b
Antagonistic activity is expressed as percentage of displacement (FSEM) of 0.4 nM of [3H]DPCPX by 10
AM of test compound. nd: not determined.
3a H H 53 (F37) 67 (F5)
3b H 3-Cl 106 (F27) 80 (F1)
3c H 4-Cl 69 (F23) 52 (F2)
3d H 3,4-Cl 57 (F36) 4 (F2)
3e 4-Cl H 132 (F21) 60 (F0)
3f 4-Cl 3,4-Cl 106 (F31) 46 (F2)
3g 3,4-Cl H 174 (F37) 51 (F0)
4 — — 14 (F27) 72 (F2)
Theophylline 15 (F7) 56 (F5)
a
Enhancing activity is expressed as percentage of decrease (FSEM) in [3H]CCPA
dissociation over control (0%) and that of PD81,723 (100%, n = 3).
b
Antagonistic activity is expressed as percentage of displacement (FSEM) of 0.4 nM of
[3H]DPCPX by 10 AM of test compound.
IV. CONCLUSION
Abbreviations
ACh Acetylcholine
cAMP Cyclic-3V,5V-adenosine monophosphate
[3H]CCPA [3H]-2-Chloro-N6-cyclopentyladenosine
CHO Chinese hamster ovary
CH3CN Acetonitrile
ACKNOWLEDGMENTS
REFERENCES
Repeat number
Location of
Disease Sites of pathology Normal Disease Protein disease (normal)a
Alzheimer’s disease Neocortex, hippocampus h Peptide; 4R, 3R tau Diffuse and senile plaques,
paired helical formation, NFT
Multiple=system tauopathy Frontotemporal regions, 4R tau NFT in oligodendroglia and
(familial) brain stem, spinal cord neurons
Progressive supranuclear Frontotemporal regions 4R tau NF NFT in astrocytes and neurons
palsy (PSP)
Corticobasal degeneration Frontotemporal regions 4R tau NFT
(CBD)
Pick’s disease Frontotemporal regions 3R tau NF Paired helical formation, NFT
Diffuse Lewy body Cerebrocortical regions, a-Synuclein Lewy bodies and neurites
disease (DLB) substantia nigra
Parkinson’s Substantia nigra, brain a-Synuclein Lewy bodies and neurites
disease nuclei
Multiple-system Cerebellum, striatal regions a-Synuclein Lewy bodies and neurites
atrophy (MSA)
Amylotrophic Brain stem, spinal cord a-Synuclein Neuronal cytoplasm
lateral sclerosis (ALS)
Familial ALS Brain stem, spinal cord SOD1 mutants Neuronal cytoplasm
Creutzfeldt–Jakob Prion protein Extracellular deposits
disease (CJD)
New variant CJD
Gerstmann–Straussler–
Scheinker disease
Fatal familial insomnia
Kuru
Figure 3 Effect of seeding and inhibitors on aggregation reaction. The lag phase
(curve c) is characteristic of reactions in which formation of nuclei for
polymerization is an unfavorable process. Addition of preformed nuclei or
‘‘seeds’’ (curve a) abolishes the lag phase. Inhibitors may affect the formation of
nuclei and influence either the lag phase, the extension of the nuclei changing the
growth phase, or both (curve d). The inhibitor example (curve d) acts more
strongly at nuclei formation than on the slope or plateau level of the growth phase.
REFERENCES
I. INTRODUCTION
In 1985 Dr. Michael Rossmann and his colleagues determined for the
first time the three-dimensional structure of a human rhinovirus [15].
Their studies, performed with human rhinovirus type 14 (HRV-14), re-
vealed the structure as an eicosahedron consisting of four proteins des-
ignated VP1, VP2, VP3, and VP4 forming a protomeric unit, combined
to form a fivefold axis of symmetry (Fig. 2). The surface of the capsid
protein contains a canyon that was shown to be the cell receptor binding
site [2]. Subsequently, the structure of several additional rhinovirus
serotypes was determined [16 –19]. Although these rhinoviruses share
the same general structure described for HRV-14, the latter appears to be
distinctly different from other rhinoviruses, particularly with respect to
the sequence similarity. Following the elucidation of the structure of
HRV-14, x-ray studies were performed on two members of the series of
compounds shown in Figure 1, disoxaril and WIN52084 [20]. The pur-
pose of this study was to elucidate the nature of the binding of these
compounds to the capsid protein.
Disoxaril was shown to bind in a hydrophobic pocket below the
a depression referred to as the ‘‘canyon,’’ with the oxazoline ring in the
‘‘toe’’ region of the binding pocket. The isoxazole ring resides in the ‘‘heel’’
below the area designated as the pore (Fig. 3). The nitrogen of the isoxazole
Figure 6 WIN52084 bound to HRV-14. The methyl group on the oxazoline ring
is pointing toward a hydrophobic pocket formed by Leu106 and Ser107.
B. Aliphatic Bridge
The x-ray studies on several analogues in this series of compounds showed
that the chain connecting the isoxazole and phenyl rings adopts a bowed
Figure 7 Plot of energy vs torsion angle from an energy profiling study resulting
from rotating the oxazoline ring of the S isomer of WIN52084 about the phenyl
ring.
Thus far all the compounds that were examined bound to HRV-14, with
the exceptions noted, are oriented with the phenyl ring in a stacking mode
with Tyr128 and Tyr152. Aromatic –aromatic interactions have been
shown to be quite common in protein – protein interactions [25 – 31],
and in many cases have displayed [32,33] an electrostatic component.
Furthermore, such interactions would be expected to contribute exten-
sively to the binding energy [34]. To determine the nature of the aromatic
stacking interactions, an energy profiling study was performed by twist-
Figure 11 Torsion angle between the oxazoline and phenyl rings obtained from
minimized structures fitted to the x-ray structure of WIN54954.
The initial x-ray study with disoxaril and WIN52084 in the binding site
revealed that asparagine 219 was within hydrogen-bonding distance of the
nitrogen of the isoxazole or oxazoline rings. Several pieces of information
suggested that this type of binding contributes negligibly if at all to the total
binding energy. Lau and Pettitt [35] examined whether the close approach
of the asparagine and isoxazole ring, which had been observed crystallo-
graphically, was indeed an attractive event. By selectively computing the
pairwise attraction of the hydrogen of the asparagine 219 and the nitrogen
of the isoxazole ring, which could conceivably be involved in the hydrogen
bond, and disregarding the contribution of this energy to the overall energy
of the system, the researchers were able to predict that the potential
hydrogen bond was inconsequential.
In addition to the computational studies that argued against the
existence of a hydrogen bond with Asn219, further evidence was obtained
by site-directed mutagenisis of the asparagine in question to an alanine
(Dr. Daniel C. Peaver, Sterling Winthrop Inc., personal communication).
Confirmation of the mutation was accomplished by sequencing. A com-
parison of the sensitivity of the mutant with the wild type showed that no
change in sensitivity had resulted from the removal of the hydrogen donor
potential. Consequently, these findings were in complete agreement with
the results reported by Lau and Pettitt.
Although the evidence presented strongly suggests the lack of con-
tribution of Asn219 to the binding energy, examination of the x-ray result
of HRV-14-bound compounds revealed the presence of a water molecule in
the vicinity of the isoxazole ring and hydrogen-bonded to the backbone of
Leu106, Ser107, and Asn219 (Fig. 12) [36]. A similar hydrogen-bonding
network has been seen in HRV-50 (Dr. Vincent Giranda, Sterling Win-
throp Inc., personal communication). This observation could shed some
light on the relative activity of other heterocyclic replacements for the
isoxazole ring.
was assumed that all the compounds included in the study that had not
been examined by x-ray crystallography behaved in a predictable manner.
Compounds were divided into two groups of seven compounds each. One
group whose conformations were known (Fig. 13) demonstrated various
levels of activity against the virus. The second group consisted of inactive
compounds with related structures (Fig. 14). In the absence of conforma-
tional data for this group, one of the active compounds was used as a tem-
plate for these compounds. A SYBYL (version 5.0) database was created.
All the structures were overlaid in the position found in the binding site
(Fig. 15). Volume maps were then calculated for the Boolean ‘‘union’’ of all
active and inactive compounds, which were then overlaid, and the excess
volume occupied by the inactive compounds, in comparison to the active
compound (Boolean minus), was calculated (Fig. 16). A similar procedure
was followed for the excess volume for the actives (Boolean plus). These
combined results revealed that inactive compounds displayed excessive
bulk around the phenyl ring. Although some bulk is desirable in this
area to enhance hydrophobic interactions, excessive bulk, which leads to
trostatic fields. Cutoffs were used to contour together points where the
correlations were highest and positive and those that were highest and
negative. Although the shapes of the maps coincide with the shape of the
pocket, the structure of the macromolecules was not part of the calcu-
lations. The visual results displayed by the contour maps qualitatively
agree with the QSAR results; that is, there is no significant correlation
between electrostatics and biological activity (Fig. 19), despite a strong
correlation between the steric fields and activity, as predicted. Although
a moderate positive effect was seen in the vicinity of the aromatic ring,
in general, this model predicts that excessive bulk in this area negatively
correlates with biological activity. These results are in agreement with the
conclusions empirically generated from the volume map study and also
confirm the lack of electrostatics involved in the phenyl–phenyl stacking
interactions, which had been observed earlier.
V. CONCLUSIONS
REFERENCES
I. INTRODUCTION
and the target amino acid sequences (at gp120 and/or gp41) with which
they putatively interact, remain to be elucidated. Most of the compounds
inhibit HIV replication at concentrations of 0.1 to 1 Ag/mL and some
(Aco-HSA and betulinic acid) are even effective within the concentration
range of 0.01 to 0.1 Ag/mL [21,22]. For the plant lectins [17,18] and mod-
ified serum albumins [20,21], the inhibitory effects on HIV replication cor-
related closely with their inhibitory effects on syncytium formation, which
corroborates the hypothesis that their anti-HIV activity is due to inhibition
of virus –cell fusion.
Whereas the plant lectins are inhibitory to HIV-1, HIV-2, and a
number of other (enveloped) viruses (viz., CMV, RSV, influenza A), at
tion in both duck hepatocytes and Pekin ducks [55]. For PMEA and
PMEDAP, but not for PMPA, PMPDAP, FPMPA, or FPMPDAP, the
activity spectrum also extends to herpesviruses (e.g., HSV, CMV). This
would make PMEA and PMEDAP particularly attractive as therapeutic
modalities in AIDS patients, since they might be useful not only for the
treatment of the underlying HIV infection but also for the therapy/
prophylaxis of the intercurrent HSV or CMV infections.
Figure 9 2V,5V-Bis-O-(tert-butyldimethylsilyl)-3V-spiro-5W-(4W-amino-1W,2W-oxa-
thiole-2W,2W-dioxide)pyrimidine (TSAO) derivatives TSAO-T, TSAO-m3T, and
TSAO-e3T.
VII. CONCLUSION
ACKNOWLEDGMENTS
REFERENCES