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inhibitor. Inhibitors binding to the primed subsites induced considerable

order in the regions 191 to 192 and 223 to 224. Most importantly, the

amide proton of His-223 formed a hydrogen bond to a carbonyl group of

the inhibitor. In addition to these changes residues remote from the

inhibitor, but near the binding sites showed increased mobility. These

results suggest that the rigidity of the S1 to S3 subsites are important for

distinguishing between ligands, while the flexible S1V to S3V subsites are

more accommodating to a broad range of residues. The flexibility of the

S1V subsite is in agreement with our observations. Interestingly, similar

studies on collagenase-1 with a hydroxymate inhibitor [19] bound to the

S1V to S3V subsites showed the analogous region to 220-226 of stromelysin1 was disordered in both the presence or absence of inhibitor. All these

results indicate that changes to mobility are complex and mostly unpre-

Figure 12 Catalytic mechanism of thermolysin and stromelysin-1. (A) The

mechanism of thermolysin [54]. (B) The mechanism of stromleysin-1 [10].

Equivalent residues to Tyr-157 and His-231 are not observed for stromelysin-1.

The proposed mechanism for collagenase-1 [53] is similar to stromelysin-1, but

also involves Asn-180 (equivalent to Asn-162 in stromelysin-1). This residue

cannot participate in stromelysin-1 due to an additional residue between Ala-165

and Asn-162. (Adapted from Ref. 10.)

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

dictable: however, such analyses may prove useful in supporting and

monitoring the presence of stabilizing interactions.

The mechanism proposed for fibroblast collagenase [53] and stromelysin-1 (10) is similar to that suggested for thermolysin [54] (Fig. 12). In

thermolysin the zinc ion ligates the carbonyl of the substrate and with Tyr157 and His-231 stabilizes the tetrahedral intermediate. In collagenase-1

and stromelysin-1, however, the stabilization of the carbonyl of the

substrate and the tetrahedal intermediate is by zinc alone. In thermolysin

the NH of the scissile bond is stabilized by a peptide carbonyl of Ala-182

and the side chain carbonyl of Asn-112. For collagenase-1 similar interactions by the peptide carbonyl of Ala-182 and the carbonyl of the side

chain of Asn-180 are suggested. For stromelysin-1, however, only the

carbonyl of Ala-165 would be involved in the stabilization of the NH of

the substrate; the equivalent Asn (Asn-162) is not involved as there is a

residue insertion in the stromelysin-1 sequence compared with the

collagenase-1 sequence. The proposed mechanisms of thermolysin, collagenase-1 and stromelysin-1 suggest that the Glu in the consensus

sequence HEXXH would promote the nucleophilic attack of water on

the scissile bond of the peptide substrate. The solution structure of

stromelysin-1 described here lacks the rigidity expected for the side chain

of this residue, Glu-202. In several members of the family of structures,

however, this side chain does approach a position that is consistent with

the mechanistic role of this residue.


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

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

additional and powerful application of NMR spectroscopy is the method

of ‘‘NMR by SAR’’ developed by Fesik et al. [59], which has been applied

to finding new drug leads for stromelysin-1 [60]. NMR spectroscopy has

clearly become a powerful and essential tool in the design and development of novel drug leads.


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Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.


Cannabinergics: Old and New

Therapeutic Possibilities

Alexandros Makriyannis

University of Connecticut, Storrs, Connecticut, U.S.A.

Andreas Goutopoulos

Serono Reproductive Biology Institute, Rockland, Massachusetts, U.S.A.



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 pharmaceuticals, 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

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

these drugs. The oily, noncrystalline nature of the biologically active

terpenoid ingredients of Cannabis sativa contributed to the lag in understanding of cannabinoid biology. The main active ingredient, (–)-delta-9tetrahydrocannabinol (delta-9-THC), was isolated and identified only in

1964 (Mechoulam, 1967), over a hundred years after the isolation of many

important crystalline biologically active natural products, such as morphine and quinine. A second reason for the lack of progress in defining the

biology of cannabimimetics was the long-standing scientific misconception

that the cannabinoid-induced pharmacological actions are mediated by

perturbation of cellular membranes rather than through specific receptors.

This hypothesis was a deterrent in the pursuit of possible specific cannabinoid binding sites. Owing to their high lipophilicity, cannabinoids were

paralleled with general anesthetics in terms of their mechanism of action

(Paton, 1975). Although cannabinoids were found to clearly perturb membranes (Makriyannis, 1987), such effects were never proven to be directly

responsible for their biological activity.

The advent of synthetic cannabimimetics with a high degree of

enantioselectivity (Johnson, 1986; Little, 1988) paved the road for the

identification of specific cannabinoid binding sites in rat brain (Devane,

1988). This discovery marked the onset of a revolution in the understanding of cannabinoid biology.


A. The CB1 Receptor

Definitive proof of the existence of the cannabinoid receptor came with

the isolation of the cDNA of a cannabinoid receptor from a rat cerebral

cortex cDNA library and its expression in Chinese hamster ovary

(CHO) cells (Matsuda, 1990). A year later, the corresponding human

receptor, named CB1, was cloned and found to share a 97.3% homology with the rat receptor (Gerard, 1991). The CB1 472 amino acid

sequence revealed (Matsuda, 1990; Gerard, 1991) that it is a member

of the G-protein-coupled receptors (GPCRs). Receptors of this family

are membrane embedded and consist of an extracellular N-terminus,

seven transmembrane helices interconnected with intra- and extracellular

loops, and an intracellular C-terminus. Bramblett et. al. (1995) constructed a model for CB1, using the known structure of bacteriorhodopsin as a starting point.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

The sites involved in interactions with G proteins of the Gi/o family

are the third intracellular loop from the N-terminal side and the Cterminus (Howlett, 1998a). The C-terminus was found to bind with high

affinity to Gi and the synthetic C-terminus peptide was found to individually stimulate GTPgS binding to G protein and to inhibit adenylate

cyclase (Howlett, 1998b). Similarly with other GPCRs, CB1 is allosterically regulated by sodium ions. It has been shown that sodium ions affect

both ligand binding and signal transduction by inducing a receptor

conformational change (Houston, 1998). There is also evidence that an

interhelical H-bonding interaction between helix II Asp and helix VII Asn

is important for the stabilization of a receptor conformational state that

has high affinity for most cannabimimetic ligands (Tao, 1998), (Howlett,

1998a). Sodium ions presumably disrupt this H bond, and thus, result in a

different, low affinity, receptor state.

The CB1 receptor is coupled with Gi (Howlett, 1998a). CB1

activation leads to inhibition of adenylyl cyclase and, therefore, to reduction of cAMP levels. Many eukaryotic cells utilize cAMP as a

second messenger that activates the cAMP-dependent protein kinase A

(PKA), which in turn phosphorylates various proteins, regulating their

function. One of the cAMP-dependent cannabinoid effects is the

enhancement of voltage-sensitive, outwardly rectifying potassium channels, which occurs as a result of decreased phosphorylation of the K+

channel protein by PKA (Deadwyler, 1995). Besides Gi, CB1 is coupled

to Go (Howlett, 1999). Furthermore, apart from inhibition of adenylyl

cyclase, CB1 utilizes several additional effector systems (intracellular

mediators) involving Gi/o proteins: the inhibition of N-type Ca2+ channels (Mackie, 1992); the activation of mitogen-activated protein kinase

(MAP kinase) (Bouabula, 1995a); and the expression of immediate early

genes like Krox-24 (Bouabula, 1995b). Other cannabinoid-induced cellular effects include activation of inwardly-rectifying potassium channels

(Pertwee, 1997) and possibly the activation of phospholipases A, C, or D

(Felder, 1995).

Different G proteins or second messengers may be coupled to CB1

in different brain regions and may mediate different physiological effects

(Howlett, 1999). Utilization of diverse effector systems by CB1 may

explain how the response to cannabimimetics varies across different cell

types. Understanding which physiological responses are mediated by

each of the foregoing intracellular signaling systems is of great significance and may suggest new approaches for the design of selective

cannabimimetic agents.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

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