A novel norindeniosoquinoline structure reveals a common interfacial inhibitor paradigm for ternary trapping of the topoisomerase I-DNA covalent complex

Abstract

We demonstrate that five topoisomerase I (Top 1) inhibitors (two indenoisoquinolines, two camptothecins, and one indolocarbazole) each intercalate between the base pairs flanking the cleavage site generated during the Top1 catalytic cycle and are further stabilized by a network of hydrogen bonds with Top1. The interfacial inhibition paradigm described for Top1 inhibitors can be generalized to a variety of natural products that trap macromolecular complexes as they undergo catalytic conformational changes that create hotspots for drug binding. Stabilization of such conformational states results in uncompetitive inhibition and exemplifies the relevance of screening for ligands and drugs that stabilize (“trap”) these macromolecular complexes.

Introduction

DNA topoisomerase I (Top1), an essential enzyme in metazoans (1, 2), has been identified as the target of camptothecin and its derivatives (3, 4). Top1 is ubiquitous and highly conserved in eukaryotes (5–7). The enzymatic activity of Top1, like that of other topoisomerases, breaks the DNA backbone reversibly by replacing a DNA phosphodiester bond with a bond between a phosphate at one end of the DNA break and a tyrosine residue of the Top1 protein. In the case of Top1 the tyrosine links to the phosphate at the 3’-hydroxyl end of the DNA break (Fig. 1A–C). DNA relaxation takes place by rotation of the free end of the broken DNA around the intact DNA strand (Fig. 1B) (8).

Figure 1.

Structure of the Top1 cleavage complex trapped by the norindenoisoquinoline AI-III-52. A–B: Top1 nicking-closing reaction. A: Top1 is generally bound non-covalently to DNA. The Top1 catalytic tyrosine (Y723 for human nuclear Top1) is represented in red (Y). A to B: Top1 cleaves one strand of the duplex as it forms a covalent phosphodiester bond between the catalytic tyrosine and the 3’- DNA terminus. The other DNA terminus is a 5’-hydroxyl (OH). B: The Top1 cleavage complex allows rotation of the 5’-terminus around the intact strand, which relaxes DNA supercoiling (purple dotted circle with arrowhead). B to A: Following DNA relaxation, Top1 religates the DNA. Under normal conditions, the religation (closing) reaction rate constant is much higher than the cleavage (nicking) rate constant. More than 90% of the Top1-DNA complexes are non-covalent (46) (as in A). C: Each of the Top1 inhibitors shown in panel F traps the Top1 cleavage complex by binding at the enzyme-DNA interface between the base pairs flanking the Top1-mediated DNA cleavage site (by convention positions −1 and +1). The colors for the base pairs −1 (light blue), +1 (dark blue) and +2 (orange) are the same as in Fig. 3. D & E: Lateral views of a Top1-DNA complex trapped by the norindenoisoquinoline AI-III-52 (shown in green and red). D: Top1 (yellow) is shown in a surface view to represent the depth of the norindenoisoquinoline binding pocket. E: Top1 is represented in ribbon diagram to allow visualization of the catalytic tyrosine (Y; red) and to show the drug intercalation between the −1 and +1 base pairs. E: Chemical structure of the five drugs co-crystallized with Top1-DNA complexes (15, 16, 24).

Camptothecin was discovered as the active principle of extracts from the Chinese tree Camptotheca acuminata by Monroe Wall and Mansukh Wani (9). Camptothecin is a planar heterocycle, consisting of 5 rings, including an important α-hydroxy-lactone E-ring (Fig. 1F). Camptothecin has several remarkable features. First, the natural alkaloid is the 20-S-camptothecin enantiomer; this is the form that is active against Top1 and experimental cancers, whereas the synthetic 20-R enantiomer lacks both activities (9). Second, camptothecin traps the Top1-DNA cleavage complex, forming a potentially lethal DNA lesion. The anticancer activity thus is not due to inhibition of the Top1 enzyme activity per se. This distinction was demonstrated in Top1-deficient yeast strains, which completely lack the camptothecin-sensitivity of wild-type strains (10–12). Third, camptothecin does not bind significantly to either DNA or purified Top1: both must be present together in the form of cleavage complexes (9). These characteristics led to the hypothesis that camptothecin forms a ternary complex with Top1 and its DNA substrate by binding to a stereospecific site in the Top1-DNA cleavage complex (13) (Fig. 1C). The DNA sequence at the cleavage sites has a strong bias for a guanine residue at the 5’ side of the DNA break (14). In view of camptothecin’s aromatic planar structure resembling a fused DNA base pair, it was hypothesized that camptothecin forms a ternary complex with Top1 and DNA, stacking against a guanine-containing base pair on the 5’ side of the break, while at the same time binding to sites on the Top1 protein (14). Camptothecin thus is stabilized at a DNA-Top1 interface in a structure similar to a normal intermediate in the topoisomerase reaction (Fig. 1C). By convention the base pairs flanking the break are referred to as −1 and +1. Position −1 corresponds to the nucleotide covalently linked to Top1 and +1 to the nucleotide at the free 5’-hydroxyl terminus (Fig. 1A–C). The atomic structure of a ternary Top1 cleavage complex was revealed by X-ray crystallography with the clinical camptothecin derivative topotecan (see structure in Fig. 1F). This structure confirmed the drug-stacking hypothesis (15). More recently, we found the binding of the natural camptothecin alkaloid to the Top1-DNA complex to be remarkably similar to the topotecan structure (16).

Because of the anticancer activity of camptothecins, many derivatives have been synthesized for clinical development (17). Two of these have been approved by the U.S. Food and Drug Administration (FDA): topotecan (Hycamtin^®) for ovarian and lung cancers and irinotecan (CPT11; Campto^®) for colon carcinomas. Several other derivatives are in preclinical development or clinical trial (Exatecan, 9-nitrocamptothecin, BAY 38–3441 (18, 19)). Camptothecins however have limitations. The alpha-hydroxylactone in the E-ring (see Fig. 1F) is rapidly converted in the circulation to a carboxylate whose tight binding to serum albumin limits the available active drug (20). Also, camptothecins are actively exported from the cell by drug efflux membrane “pumps” (21).

Non-camptothecin Top1 inhibitors are therefore of great pharmaceutical interest to overcome the limitations of camptothecins. Moreover, because drugs from different chemical families that share the same molecular target generally have different spectra of clinical activity, non-camptothecin Top1 inhibitors may be effective against different tumors. For instance, this is the case for the tubulin and the topoisomerase II inhibitors (17). Indenoisoquinoline and indolocarbazole derivatives (Fig. 1F) are being pursued for therapeutic development. The first indenoisoquinoline Top1 inhibitor was discovered by searching the NCI cell screen database for compounds resembling camptothecin in their cytotoxicity profile against 60 human cancer cell lines (22). In the absence of definitive structural information of the drug target site, derivatives were initially designed with amino groups that would bind electrostatically to the polyanionic phosphodiester backbone. Over 300 indenoisoquinoline derivatives have been synthesized to date, and several potent and selective Top1 inhibitors have been identified as potential anticancer agents (17, 23). We recently reported the first crystal structure of an indenoisoquinoline (MJ-238) (Fig. 1F) bound to a Top1 cleavage complex (16). In the same study, we also reported the structure of the natural camptothecin alkaloid and of an indolocarbazole (SA315F) (Fig. 1F) bound to the Top1-DNA complex (16).

This study describes the crystal structure of a potent norindenoisoquinoline (24) Top1 inhibitor (AI-III-52) in a complex with DNA and Top1. Comparison with the corresponding ternary complexes of the MJ-238 indenoisoquinoline (16), and with the structures of camptothecin (16), topotecan (15) and of an indolocarbazole (16), reveals a common molecular mechanism of drug action. These Top1 inhibitors all bind at the Top1-DNA interface by intercalating and stacking between the base pairs flanking the DNA cleavage site and by forming critical hydrogen bonds with Top1 amino acid residues. These same Top1 residues have been implicated previously in enzyme catalysis and/or resistance to camptothecins (25). We discuss Top1 inhibitors as paradigms for drugs that trap macromolecular interfaces, and the generality and implications of the interfacial inhibitor concept (26, 27).

Materials and Methods

A 70 kiloDalton construct of human topoisomerase I (top70), residues Lys175 to Phe765, was expressed and purified from baculovirus-insect cells (SF9) as described previously (28). Crystallization trials were conducted with wild-type topo 70 and duplex DNA oligomers containing a 5' bridging phosphorothiolate linkage at the site of DNA cleavage (15, 16). Crystals were grown by sitting drop vapor diffusion by preparing drops containing 2 ml precipitant, 1.5 ml of 50 mM duplex DNA, 0.5 ml compound and 1.5 ml of 4.2 mg/ml protein. Precipitant was 10–12% PEG 8000, 100 mM MES pH 6.4, 200 mM lithium sulfate. The compound solutions used were 1–5 mM of ligand dissolved in 0–10% DMSO v/v in water. Crystals were cryo-protected for data collection by passing them through precipitant plus 30% v/v PEG 400. Data were collected at 100 K at Beamline 5.0.3, Advanced Light Source, Lawrence Berkeley National Laboratory. Structures were solved by molecular replacement using the program AMORE (29) with protein coordinates from the top70 ternary complex structure with topotecan (15). Refinements were conducted using CNX (30) and REFMAC (31) and iterative model adjustments with XtalView (32) (see Table 1). DNA’s were placed into the |Fo|-|Fc| electron density and refined. Drug molecules were then placed into the to |Fo|-|Fc| electron density using QUANTA module X-LIGAND (33). The diffraction resolution of the crystals obtained was 3.1 Å and this did not allow the placement of water molecules.

Table 1.

Refinement Statistics^b

Inhibitor bound	AI-III-52
PDBID	1TL8
Resolution (Å)	50-3.1 (3.21-3.10)
No. reflections	16738 (1581)
Rsym a	10.0 (52.2)
Completeness	97.8 (93.1)
I/σI	13.7 (2.3)
Space Group	P21
a (Å)	56.949
b (Å)	114.140
c (Å)	73.499
β	94.18
Reflections used in RFREE	5%, 808
No. of protein atoms	4703
No. of DNA atoms	892
No. of inhibitor atoms	24
No. of solvent atoms	0
RFACTOR	22.9 (33.2)
RFREE	30.5 (39.5)
r.m.s. deviations from ideal stereochemistry
bond lengths (Å)	0.017
bond angles (°)	1.9
impropers (°)	3.94
dihedrals (°)	23.8
Mean Bfactor –all atoms (Å3)	60.4

^aR_sym=Σ |I_i − I_m|/Σ I_m where I_i is the intensity of the measured reflection and I_m is the mean intensity of all symmetry related reflections.

^bNumbers in parenthesis represent final shell of data.

Molecular visualization was performed using PyMOL. The PDB codes for the different Top1 crystal structures were 1LT8 for norindenoisoquinoline (24); 1SC7 for MJ-238 (16); 1SEU for indolocarbazole (16); 1K4T for topotecan (15); 1T81 for camptothecin (16) and 1A31 for Top1 in the absence of inhibitor (34).

Results and Discussion

Norindenoisoquinoline intercalates between the base pairs flanking the Top1-mediated DNA break

The overall structure the AI-III-52 norindenoisoquinoline in a Top1 cleavage complex is presented in Figure 1D & E. The drug is bound deep inside the Top1 (Fig. 1D), intercalated between the base pairs flanking the DNA cleavage site (positions −1 and +1) (Fig. 1E). The Top1 protein encircles the DNA in a closed clamp conformation (34–36). The polypeptide hinge is behind the DNA and the tight narrow cleft through which the DNA has presumably entered the enzyme is represented in front of the DNA. The Top1 enzyme used in these experiments comprises the core, linker and catalytic domain. It does not include the Top1 N-terminal domain that encompasses residues 1–174 and which is not well conserved among eukaryotic Top1 enzymes. The N-terminus has been shown both in vitro and in vivo to be dispensable with respect to the biochemical and cytotoxic effects of Top1 poisons including camptothecin and topotecan (28, 37). Technical aspects of structure determination are summarized in Table 1.

Figure 2A shows two expanded views of AI-III-52 bound to the DNA in the Top1 cleavage complex. The left panel is in the same view orientation as Figure 1D & E with the DNA cleavage site seen from the minor groove. The right panel is a 90° rotation showing AI-III-52 under the +1 nucleotide pair. The +1 base pair (shown as capped sticks) covers and stacks against the entire drug (shown in space filling model). Extensive stacking is also the case for the −1 base pair, which is completely covered by the drug and therefore not shown in Fig. 2A-right. Figure 3B shows the relative positions of the base pairs flanking the intercalated norindenoisoquinoline molecule. The +1 base pair is oriented as in Figure 2A-right, and the drug molecule is not shown. The longitudinal axes of the +1 and −1 base pairs (represented by thin dashed lines) are almost parallel. Thus, the twist angle between the base pairs flanking the intercalated drug is reduced to 10°.

Figure 2.

Stacking of a drug molecule between two base pairs flanking the Top1 cleavage complex is a common mechanism for the indenoisoquinolines (A, B), the camptothecins (C, D) and an indolocarbazole derivative (E). Each panel shows 2 views for each drug within the Top1 cleavage complex. The left views are oriented as in Fig. 1 D & E with the DNA viewed from the minor groove. The right views are rotated 90° and show the +1 nucleotides covering the drug molecules. In each panel, the catalytic Top1 tyrosine is shown in red (Y) at the top left of each left view. The −1 and +1 base pairs are marked by dashed arrows. In the right views, the colored numbers correspond to the drug atoms numbered in Figure 4. The orientation of SA315F is the same in the middle and right panels in Figure 2E.

Figure 3.

DNA unwinding by the norindenoisoquinoline AI-III-52. A: Twist angle between the base pairs flanking the Top1 cleavage site in the absence of inhibitor (from (34)). The thin dashed lines correspond to the base pair long axes. The +1 base pair is colored dark blue and the −1 base pair cyan. B: Twist angle between the base pairs flanking the Top1 cleavage site trapped by AI-III-52. The drug has been removed to only show the −1 and +1 nucleotides. The +1 nucleotides are positioned similarly to panel A. The thin dashed lines correspond to the base pair long axes. C: Twist angle between the +1 and +2 base pairs. The color code is the same as in Fig. 1A–C.

DNA unwinding is a characteristic of DNA intercalators, caused by the increased distance between flanking base pairs required to accommodate the intercalated molecule. For instance, intercalation of ethidium reduces the twist angle between the flanking bases pairs to 7°, compared to the normal 33°. The reduction in the twist angle between flanking base pairs for the norindenoisoquinoline AI-III-52 intercalated in the Top1-DNA cleavage complex (Fig. 3B) is comparable to that produced by intercalated ethidium. The twist angle reduction observed with AI-III-52 is not due to Top1 cleavage, because the twist angle between the base pair flanking the Top1 cleavage site in the absence of drug remains near normal (Fig. 3A) (34). The twist angle of 58 ° between the +1 and +2 base pairs in the norindenoisoquinoline structure indicates slight overwinding of the DNA immediately adjacent to the drug binding (Fig. 3C). This overwinding probably compensates for the unwinding produced by the norindenoisoquinoline.

Camptothecin, topotecan, MJ-238 and indolocarbazole also intercalate between the base pairs flanking the Top1-mediated DNA break

Figure 2B shows that the indenoisoquinoline MJ-238 (see structure in Fig. 1F) like the norindenoisoquinoline AI-III-52 stacks and intercalates between the base pairs −1 and +1 on each side of the cleavage site generated by Top1. For both drugs, their aromatic portion is parallel to the −1 and +1 base pair long axes. However, the two indenoisoquinolines are flipped 180° (Fig. 2 and Fig. 4, compare panels A & B). Numbering of the corresponding atoms makes this difference apparent. For AI-III-52, the sequence of atoms in a clockwise orientation is 1, 10 and 6, whereas it is 1, 6 and 10 for MJ-238. In other words, AI-III-52 is intercalated with the nitrogen N6 in the minor groove. This nitrogen N6 can hydrogen bond with the Top1 R364 residue. By contrast, MJ-238 has the nitrogen N6 in the major groove of the DNA and the C11 carbonyl hydrogen-bonded to R364. The side chain attached to the nitrogen N6 protrudes in the DNA major groove (Fig. 2B).

Figure 4.

Hydrogen bond networks between drugs and Top1 amino acid residues in the drug-Top1-DNA ternary complexes. A: AI-III-52 forms two direct hydrogen bonds with Asn722 and Arg364. B: MJ-238 forms only one hydrogen bond with Arg364 (16). C: The natural camptothecin forms 3 hydrogen bonds with Asp533, Asn722 and Arg364 (16). D: Two hydrogen bonds have been reported for topotecan with Asn722 and Arg364 (15). The third hydrogen bond with Arg364 has been proposed (see Discussion). Note that the water-mediated Asp722 hydrogen bond is represented with the amino group of N722. It is also plausible to be with the carbonyl of N722 (not shown).

Like the indenoisoquinolines, the two camptothecins intercalate between the −1 and +1 base pairs at the cleavage site of the DNA-Top1 complex (15, 16) (Figs. 2C–D). Both camptothecins stack in the same orientation. Their 5-membered planar heterocyclic structure coincides with the long axis of the base pairs, and their shapes closely match that of a base pair. The hydroxyl substituents at the 20-position are in close proximity with the sugar of the −1 nucleotide and the DNA phosphodiester backbone and contribute to the optimum stacking of the natural 20S-camptothecin enantiomer. This explains why the 20R- camptothecin enantiomer cannot trap the Top1 catalytic complex (13). At the other end of the camptothecin, the A ring is not immediately in contact with the DNA phosphodiester backbone opposite from the cleavage site. This space can accommodate the 10-hydroxy substituent of topotecan. Filling this space probably accounts for the greater potency of 10-hydroxy-substituted camptothecins and for the even greater activity of 10- 11-dimethoxy-camptothecin derivatives (13).

The indolocarbazole SA315F is also bound with maximum stacking interactions. The indolocarbazole aromatic portion is aligned with the axis of the −1 and +1 base pairs (16) (Fig. 2E). The deoxyribose is positioned in the major groove.

For the indenoisoquinoline MJ-238, topotecan, and the indolocarbazole SA315F, the drug aromatic polycyclic ring is parallel to the +1 base pair axis and the bulky substituents encroach the major groove. This common orientation would suggest that drug substitutions that impinge on the minor groove may be disfavored. Consistent with this possibility, substitutions on the camptothecins at positions 1 and 12 (see Fig. 1F) preclude Top1 inhibition (13). Also, minor groove substitutions with benzo[a]pyrene diol epoxide adducts on deoxyguanine N2 prevent Top1 from cleaving DNA when they occupy the Top1 site in the DNA minor groove (38). In contrast, substitutions towards the minor groove on indenoisoquinolines at position 11 (see Fig. 1F) result in more potent compounds for cytotoxity and Top1 inhibition (39–41). This suggests that these substitutions directly interact with Top1 residues and strengthen the hydrogen bond network for these particular compounds.

The indenoisoquinolines and the camptothecins bind to Top1 by a common hydrogen bond network

A schematic representation of the hydrogen bond network for the two indenoisoquinolines and the two camptothecins is shown in Figure 4. Two of the Top1 catalytic residues are shown. Y723 forms the covalent bond with DNA. K532 serves a dual function. K532 forms a hydrogen bond on the minor groove side with the −1 base on the scissile strand (8, 15) and probably also functions as a general acid during cleavage to protonate the leaving 5’ oxygen (42). Three other Top1 residues form hydrogen bonds with the intercalated drugs: N722, R364, and D533. None of these residues are essential for Top1 catalytic activity. However, each of them has been implicated in resistance to camptothecins (4), indolocarbazoles (43), and indenoisoquinolines (23).

The norindenoisoquinoline AI-III-52 forms two hydrogen bonds, one with R364 and one with N722 (Fig. 4A). Only one hydrogen bond was detected for MJ-238 (16): with R364 (Fig. 4B). The weaker Top1 inhibitory activity of MJ-238 compared to AI-III-52 (16, 24) can therefore be attributed to its reduced hydrogen bonding with Top1 and to reduced stacking because of the smaller surface of its aromatic portion (compare panels A and B in Fig. 2 and Fig. 4).

The two camptothecins have a more similar H-bond network with the Top1-DNA complex than the indenoisoquinolines (Fig. 4). For camptothecin three hydrogen bonds have been reported (16) (Fig. 4C). Two are direct. One is between the camptothecin 20-hydroxy and D533, and the other between the camptothecin N1 and R364. The third H-bond has been proposed to be a water-mediated between the 17-carbonyl on the camptothecin D-ring and N722 of Top1 (16). For topotecan, only two of the three H-bonds (with D533 and N722) were initially reported (15) and are shown in Figure 4D. Reexamination of the topotecan ternary structure reveals that the R364 side chain is also in close proximity with N1 of topotecan and is positioned for hydrogen-bonding (15).

Generalization of the interfacial inhibitor concept

To our knowledge camptothecins were the first inhibitors hypothesized (14, 44) and demonstrated (15) to bind at the “interface of two macromolecules” (Top1 and its DNA substrate) as these macromolecules undergo a catalytic conformational change (the “cleavage complex” – see Fig. 1A–C). This mechanism can be generalized as it also applies to three non-camptothecin inhibitors: two indenoisoquinolines and an indolocarbazole (see Fig. 2). The implications of these structures are several folds. First, they will aid for the rational synthesis of novel Top1 inhibitors. However, the example of the two indenoisoquinolines that bind in flipped orientation (see Fig. 2A and B) demonstrate the potential limitations of simple extrapolations from crystal structures, and the need to correlate structural biology predictions with biological testing and ultimately additional atomic resolutions; hence the need of an iterative process from chemistry to biology to structural biology, and again.

A second implication of the interfacial binding of Top1 inhibitors is its apparent generality. The “interfacial inhibition” paradigm can be extended to a wide range of drugs and macromolecular targets (Table 2) (26, 27). We therefore define “Interfacial Inhibitors” as drugs that bind at the interface of two (or more) macromolecules as the multimeric complex undergoes a structural transition. These multimeric complexes can present a drug binding site at their protein-protein interface as in the case of brefeldin A and tubulin inhibitors (colchicine, vinblastine, paclitaxel [Taxol®]) or a drug binding site at nucleic acid-protein interfaces as in the case of Top1 inhibitors (present study), the RNA polymerase II inhibitor α-amanitin and the antibiotic ribosome inhibitors (Table 2 (26, 27), and Fig. 3 in a recent review (27)).

Table 2.

Natural products that act as interfacial inhibitors

Drug	Origin	Substrate	Mechanism	Refs.
Camptothecin	Chinese tree	Topoisomerase I- DNA complex	Stabilizes a kinetic intermediate of the DNA cleavage enzymic reaction.	(15, 25, 47)
Brefeldin A	Ascomycetes	Arf-GDP-GEF complex	Stabilizes a kinetic protein-protein intermediate undergoing conformational changes.	(48, 49)
Cyclosporine	Soil Fungus	Cyclophilin/ Calcineurin	Hinders access to the active site of the protein phosphatase calcineurin by artificially creating a protein- protein interface	(50, 51)
FK506/ tacrolimus	Actinomycetes (fungi-like bacteria)	FKBP/Calcineurin	Idem	(52, 53)
Forskolin	Indian herb	Heterodimer of adenylyl cyclase	Activates the enzyme by stabilizing the heterodimer catalytic site in an active conformation	(54)
Fusicoccin	Fungus (plant parasite)	14-3-3/ATPase complex	Overstabilizes a regulatory complex	(55)
Rapamycin	Soil bacteria	FKBP/FRAP	Promotes dimerization of FKBP12 with FRAP and inhibits mTOR	(56)
Colchicine	Crocus	Tubulin heterodimer	Prevents tubulin polymerization by stabilizing a curved tubulin heterodimer	(57)
Vinblastine	Vinca	Tubulin heterodimer	Prevents tubulin polymerization by stabilizing a curved tubulin heterodimer	(58)
Taxol	Tree (Taxus baccatus)	β-tubulin	Stabilizes microtubules	(59)
Epothilone A	Myxobacteria	β-tubulin	Stabilizes microtubules	(60)
Kirromycin	Fungus	Antibiotics: bacterial ribosomes	Stabilization of translation elongation factors EF-Tu	(61, 62)
Fusidic acid	Fungus	Bacterial ribosome	Stabilization of translation factor EF- G	(61, 62)
Thiostrepton	Streptomyces	Bacterial ribosome	Prevents EF-G binding by altering the interface between ribosomal protein L11 and rRNA	(63)
Dexrazoxane (= ICRF-187)	Synthetic	Topoisomerase II-ATP complex	Stabilizes the closed topoisomerase II dimer	(64, 65)
α-Amanitin	“Black cap” mushroom	RNA polymerase II	Binds to Rpb1-Rpb2 interface and prevents Pol II translocation	(66)

Interfacial inhibition is reversible and uncompetitive (26, 27). Interfacial inhibitors bind to and trap a catalytic intermediate of the macromolecular complex in a specific configuration (Fig. 5A). Interfacial inhibition represents a paradigm for “uncompetitive inhibition” (Fig. 5B and C: Lineweaver-Burke plot equations). The pharmacological activity of interfacial inhibitors is due, at least in part to the conversion of the trapped macromolecular intermediate into lethal lesions by other macromolecules that interact with the normally transient ternary drug-macromolecular complex. For instance, in the case of Top1 inhibitors, the cleavage complexes are only lethal when they are converted into replication double-strand breaks by the collision of a DNA polymerase with a Top1 cleavage complex reversibly trapped by the drug (45). This explains why the antiproliferative activity of Top1 inhibitors increases with the levels of Top1. Conversely, a common resistance mechanism to Top1 inhibitors is downregulation of Top1 (4). Hence interfacial inhibitors differ from competitive inhibitors. Increasing drug target increases resistance to competitive inhibitors whereas it sensitizes to interfacial inhibitors.

Figure 5.

Generalization of the interfacial inhibitor paradigm. A: Schematic cartoon representing the interfacial inhibition paradigm applied to an enzymatic system: 1) A substrate (S) binds to an enzyme (E); 2) The enzyme converts the substrate to product (S - > P); 3) P dissociates and the enzyme is regenerated for a new catalytic cycle; 4) The interfacial inhibitor (I) binds at the enzyme-substrate "interface" and traps the enzyme-substrate complex, thereby reversibly preventing conversion of S to P. B: Equilibrium representation of interfacial inhibition. K_S: substrate equilibrium dissociation constant; K_I: inhibitor equilibrium dissociation constant; k: catalytic constant. C: Equations corresponding to uncompetitive scheme shown in panel B. V: reaction velocity; K_M: Michaelis-Menton equilibrium dissociation constant (K_M = K_S when k is rate limiting).

The interfacial inhibitor paradigm has implications for drug discovery as interfacial inhibitors stabilize rather than inhibit the formation of macromolecular complexes. Assays should therefore be designed to look for drugs that stabilize rather than inhibit the formation of macromolecular complexes. Interfacial inhibitor screening using various methods to measure the binding of macromolecules has the potential to discover highly selective inhibitors for one particular receptor site and/or enzyme substrate.