Molecular modelling and drug design.

Drug design is a creative act of the same magnitude as composing, sculpting, or writing. The results can touch the lives of millions, but the creator is rarely one scientist and the rewards are distributed differently in the arts than in the sciences. The mechanisms of creativity are the same, i.e., incremental (plodding from darkness to dawn) or sudden (the "Eureka" effect) realization, but both are poorly understood. Creativity remains a human characteristic, but it is directly related to the tools available, especially computer software and hardware. While modelling software continues to mature, very little new has evolved in terms of hardware. Here, we discuss the history of molecular modelling and describe two novel modelling tools, a haptic device and a program, SCULPT, to generate solid molecular models at atomic resolution.

Crystal structures of the complex of porcine pancreatic elastase with two valine-derived benzoxazinone inhibitors.

The crystal structures of porcine pancreatic elastase complexed to two similar benzoxazinone inhibitors are reported to 2.09 A and 1.76 A resolution, and refined to conventional R factors of 0.153 and 0.172.

Structure of the product complex of acetyl-Ala-Pro-Ala with porcine pancreatic elastase at 1.65 A resolution.

A single crystal of porcine pancreatic elastase was mounted in a thin-walled capillary and allowed to react with acetyl-Ala-Pro-Ala-paranitroanalide. Diffraction data to 1.65 A resolution were measured and the isomorphous structure was solved from the difference Fourier map. The structure contains two surprises. Two molecules of the product: acetyl-Ala-Pro-Ala molecule are bound in the extended binding site. Both molecules are bound backwards with respect to the established mode of peptide binding.

Stereochemistry of binding of the tetrapeptide acetyl-Pro-Ala-Pro-Tyr-NH2 to porcine pancreatic elastase. Combined use of two-dimensional transferred nuclear Overhauser enhancement measurements, restrained molecular dynamics, X-ray crystallography and molecular modelling.

A nuclear magnetic resonance study of the conformation of the tetrapeptide acetyl-Pro-Ala-Pro-Tyr-NH2 bound to porcine pancreatic elastase is presented. From two-dimensional transferred nuclear Overhauser enhancement measurements, a set of 23 approximate distance restraints between pairs of bound ligand protons, indicative of an extended type structure, is derived. The structure of the bound tetrapeptide is then refined from two different starting structures (an extended beta-strand and a polyproline helix) by restrained molecular dynamics, in which the interproton distances are incorporated into the total energy of the system in the form of effective potentials. Convergence to essentially the same average restrained dynamics structures is achieved. The refined structures are then modelled into the active site of elastase by interactive molecular graphics. The determination of the anchor point of the bound tetrapeptide on the enzyme was aided by a simultaneous crystallographic study which, despite the fact that only electron density for a Pro-X dipeptide fragment was visible, enabled both the approximate position and orientation of binding to be determined. It is found that the tetrapeptide is bound in the S' binding site in the reverse orientation found in other serine protease-inhibitor complexes and is stabilized both by hydrogen-bonding and by van der Waals' interactions.

X-ray diffraction analysis of the inhibition of porcine pancreatic elastase by a peptidyl trifluoromethylketone.

X-ray crystallographic data to 2.57 A resolution (1 A = 0.1 nm) have been measured for the complex of a peptidyl trifluoromethylketone inhibitor with porcine pancreatic elastase (PPE); R = 0.14. The inhibitor forms a stable complex with the enzyme by means of a covalent attachment to active site Ser195O gamma, resulting in a hemiketal moiety with tetrahedral geometry. The tripeptide protion binds as an antiparallel beta-sheet, with four hydrogen bonds augmenting the active-site covalent linkage, Ki = 9.5 microM. His57 exhibits a bifurcated H-bond to both Ser195O gamma and an F atom of the inhibitor. This study is one of a series which explores the binding geometry of a variety of small substrates and inhibitors to PPE. This peptidyl-PPE complex affords insight into the binding geometry of a novel trifluoromethylketone moiety to a serine proteinase.

Structure of recombinant mouse collagenase-3 (MMP-13).

The matrix metalloproteinases are crucial in the physiological and pathological degradation of the mammalian extracellular matrix, including breast tumours, and osteoarthritic cartilage. These enzymes are classified according to their matrix substrate specificity. Collagenase-3 (MMP-13) is a member of this family and preferentially cleaves type II collagen, cartilage, fibronectin and aggrecan. Collagenase-3 is normally expressed in hypertrophic chondrocytes, periosteal cells, and osteoblasts during bone development. The structure of the catalytic domain of recombinant mouse collagenase-3, complexed to the hydroxamate inhibitor (RS-113456), is reported at 2.0 A resolution. Molecular replacement and weak phasing information from a single derivative determined the structure. Neither molecular replacement nor derivative methods had a sufficient radius of convergence to yield a refinable structure. The structure illuminates the atomic zinc ion interactions with functional groups in the active site, emphasizing zinc ligation and the very voluminous hydrophobic P1' group for the inhibitor potency. The structure provides insight into the specificity of this enzyme, facilitating design of specific inhibitors to target various diseases.

The structure of an insect chymotrypsin.

The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme.

Structures of adamalysin II with peptidic inhibitors. Implications for the design of tumor necrosis factor alpha convertase inhibitors.

Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor alpha convertase, TACE, involved in the release of the inflammatory cytokine, TNFalpha. The structure of adamalysin II in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 A resolution. The inhibitors bind to the S'-side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded beta-sheet and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin-based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors.

Effect of the 7-amino substituent on the inhibitory potency of mechanism-based isocoumarin inhibitors for porcine pancreatic and human neutrophil elastases: a 1.85-A X-ray structure of the complex between porcine pancreatic elastase and 7-[(N-tosylphenylalanyl)amino]-4-chloro-3- methoxyisocoumarin.

A series of new acyl, urea, and carbonate derivatives of 7-amino-4-chloro-3-methoxyisocoumarin were synthesized and evaluated as irreversible inhibitors of human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE). Inhibition of HNE is directly related to the hydrophobicity of the substituent on the 7-amino group. The N-Tos-Phe derivative (19) is the best HNE inhibitor with a second-order rate constant kobs/[I] = 200,000 M-1 s-1. The closest analogue in this series, the 3,3-diphenylpropionyl derivative 5, had a kobs/[I] = 130,000 M-1 s-1 with HNE. In contrast to the Tos-Phe derivative 19, phenylacetyl derivative 2 and carbonates 22 and 25 gave extremely stable enzyme-inhibitor complexes with deacylation half-lives longer than 48 h with both elastases. N-Phenylurea derivative 25 was the best inhibitor for PPE with a second-order rate constant kobs/[I] = 7300 M-1 s-1. The crystal structure of a complex of PPE with N-tosyl-Phe derivative 19 was determined at 1.85-A resolution and refined to a final R factor of 16.9%. The isocoumarin forms an acyl enzyme with Ser-195, while His-57 is near the inhibitor, but not covalently linked. The Tos-Phe makes a few hydrophobic contacts with the S' subsites of PPE, but appears to be interacting primarily with itself in the PPE structure. This region of HNE is more hydrophobic and modeling indicates that the inhibitor would probably make additional contacts with the enzyme.

Simulations of dynamical properties of a Michaelis complex: porcine pancreatic elastase and the hexapeptide, Thr-Pro-n Val-Leu-Tyr-Thr.

Molecular dynamic simulations (30ps) of the Michaelis complex of hexapeptide (Thr-Pro-nVal-Leu-Tyr-Thr) bound to porcine pancreatic elastase (PPE) hydrated by about 2000 water molecules have been performed using the AMBER 3.0 program package. Dynamical properties of the conformation of the active site have been examined. A comparison with previously reported simulations of native PPE shows that after the substrate is bound, the catalytically crucial H-bond between O gamma-H group of (Ser 195) and nitrogen N epsilon (His 57) is more readily formed. These results show, however, that the H-bond does not adopt the most favorable conformation. The O gamma-H group of Ser 195 has a statistical preference for an attractive interaction with the O = C carbonyl (Ser 214) rather than the nitrogen N epsilon (His 57).

Structure-activity relationships of sulfonamide drugs and human carbonic anhydrase C: modeling of inhibitor molecules into the receptor site of the enzyme with an interactive computer graphics display.

We have analyzed the molecular interaction of 28 sulfonamide inhibitors with human carbonic anhydrase C (HCAC) using an interactive computer graphic display. Small aromatic sulfonamides gain most of their inhibitory power towards HCAC from the interaction of hydrogen bond acceptors at the para or meta positions with hydrophilic residues of the enzyme. Additional coordinated water molecules stabilize the complexes of heterocyclic sulfonamides (i.e., thiophenes, 1,3-thiazoles, and 1,3,4-thiadiazoles) with HCAC. We propose two different modes of binding of the heterocyclic ring with respect to the active site cleft: the heterocyclic sulfur atom of a 3,4-unsubstituted thiophene approaches the oxygen atom of a coordinated water molecule (sulfur "out"), whereas in 3,4-unsubstituted 1,3,4-thiadiazoles, the sulfur is in contact with a hydrophobic part of the receptor site (sulfur "in"). This proposal is consistent with crystallographic evidence. Sulfonamides with two aromatic or heterocyclic rings also interact with a hydrophobic pocket of the enzyme located greater than 10 A away from the active site metal Zn2+. We also discuss the possibility that the relative inactivity of thiazide diuretics is due to the steric interaction of the ortho chlorine atom with the enzyme receptor cavity.

The crystal structure of adamalysin II, a zinc-endopeptidase from the snake venom of the eastern diamondback rattlesnake Crotalus adamanteus.

1. Adamalysin II, alias proteinase II, a 24-kDa zinc-endopeptidase from the snake venom of Crotalus adamanteus, is a member of a large family of metalloproteinases isolated as small proteinases or proteolytic domains of mosaic hemorrhagic proteins from various snake venoms. Homologous domains have been recently detected in multimodular mammalian reproductive tract proteins and in mammalian gene products, somatic rearrangements of which seem to be linked to primary breast cancers. 2. The 2.0 A X-ray crystal structure of adamalysin II reveals an ellipsoidal molecule with a shallow active-site cleft separating a relatively irregularly folded sub-domain from the main molecular body composed of a 5-stranded beta-sheet and four alpha-helices. Opposite to this active-site cleft is an integrated calcium ion liganded by carbonyl and strongly conserved carboxylate/carboxamide residues. The folding of the peptide fragment containing the zinc-binding motif HExxHxxGxxH bears only a distant resemblance to thermolysin; it is identical to that found in astacin, in collagenases, and in serralysins, with the three histidines (His142, His146, His152) and a water molecule (linked to the glutamic acid Glu143) likewise constituting the zinc ligand; similar to collagenases, but in contrast to astacin, adamalysin II lacks a fifth (tyrosine) zinc ligand, leaving its zinc-ion tetrahedrally coordinated. Furthermore, adamalysin II shares an identical active-site basement formed by a common Met-turn. 3. Due to their virtually identical active-site environment and similar folding topology, the snake venom metalloproteinases (hitherto called adamalysins) and the three other proteinases might be grouped into a common superfamily called metzincins with distinct differences from the thermolysin family.

Energy minimization and molecular dynamics studies of Asn-102 elastase.

Four isomeric forms of the Asn-102 PPE (D102N mutant according to the emerging protocol, [Knowles, Science, 236 (1987) 1252-1258]) have been investigated using energy minimization (EM) and molecular dynamics (MD) techniques. MD simulation data for 175 ps are reported for each form (in total 700 ps for about 2500 atoms). The His-57 N epsilon-protonated forms are calculated to be more stable than the N delta-protonated ones. The active site region of the most stable form is very similar to that found in the D102N rat trypsin enzyme [Craik et al., Science, 237 (1987) 909-913]. Conformations of the active sites and their hydrogen bond patterns are presented for each of these forms and are compared with the structure of the native enzyme active site. The pH dependent activity of the D102N derivative is discussed.

Thermodynamic cycle-perturbation study of the binding of trifluoroacetyl dipeptide anilide inhibitors with porcine pancreatic elastase.

The variety of results of crystallographic studies of the serine proteases complexed with isocoumarin inhibitors presents a challenging problem to modeling methods and molecular energetics. Therefore, the thermodynamic cycle-perturbation technique has been used to study a model system of elastase and two peptidic inhibitors. Using the program AMBER, the technique correctly predicts changes of the binding constants for the trifluoroacetyl dipeptide inhibitors in comparison with available experimental (kinetic and crystallographic) data. However, the absolute values obtained are shown to be sensitive to the specific electrostatic interaction potential parameters used in the simulations. The reader and user are cautioned that thermodynamic cycle-perturbation results may be too optimistic by underestimating the accuracy of free energy values. This is especially a matter of concern for those cases where a direct comparison with experimental values is not possible, viz., (1) the stimulation of binding of novel compounds, (2) structurally uncertain binding sites, or (3) structurally different binding modes. With our best 4-31G* ESP (electrostatic potential) charges we were able to reproduce experimentally determined free energy differences (delta delta A) with an accuracy of about 1.5 kcal/mol. Dynamically induced structural changes in the binding site of elastase, and particularly changes in hydrogen-bond patterns of the binding site, are also reported.

Computer aided prediction and evaluation of the tertiary structure for rat elastase II.

Predictive methods have been extended to generate a proposed tertiary structure of rat elastase II on the basis of primary amino acid sequence and structural homologies within the family of mammalian serine proteinases. Force field refinement calculations were used to relax the structure. Structurally conserved molecules of solvation were introduced and the structure was again refined by means of force-field calculations. The resulting structure suggests probable substrate cleavage preferences. An independent statistical analysis of the crystallographically refined structures of serine proteinases (0.01-0.13 A, RMS) shows a close similarity to the final predicted model of rat pancreatic elastase II (0.03-0.14 A, RMS).

Acetazolamide or dexamethasone use versus placebo to prevent acute mountain sickness on Mount Rainier.

Eighteen climbers actively ascended Mount Rainier (elevation 4,392 m) twice during a randomized, double-blind, concurrent, placebo-controlled, crossover trial comparing the use of acetazolamide, 250 mg, dexamethasone, 4 mg, and placebo every 8 hours as prophylaxis for acute mountain sickness. Each subject was randomly assigned to receive placebo during one ascent and one of the active medications during the other ascent. Assessment of acute mountain sickness was performed using the Environmental Symptoms Questionnaire and a clinical interview. At the summit or high point attained above base camp, the use of dexamethasone significantly reduced the incidence of acute mountain sickness and the severity of symptoms. Cerebral and respiratory symptom severity scores for subjects receiving dexamethasone (0.26 +/- 0.16 and 0.20 +/- 0.19, respectively) were significantly lower than similar scores for both acetazolamide (0.80 +/- 0.80 and 1.20 +/- 1.05; P = 0.25) and placebo (1.11 +/- 1.02 and 1.45 +/- 1.27; P = .025). Neither the use of dexamethasone nor that of acetazolamide measurably affected other physical or mental aspects. Compared with placebo, dexamethasone appears to be effective for prophylaxis of symptoms associated with acute mountain sickness accompanying rapid ascent. The precise role of dexamethasone for the prophylaxis of acute mountain sickness is not known, but it can be considered for persons without contraindications who are intolerant of acetazolamide, for whom acetazolamide is ineffective, or who must make forced, rapid ascent to high altitude for a short period of time with a guaranteed retreat route.

Crystallographic analysis of the inhibition of porcine pancreatic elastase by a peptidyl boronic acid: structure of a reaction intermediate.

The crystal structure of porcine pancreatic elastase (PPE) complexed to carbobenzoxy-alanylisoleucine-boronic acid (ZAIB) is reported to 2.09-A resolution and refined to an R factor of 0.15. This is the first reported structural analysis of PPE with an isoleucine residu in the primary specificity pocket. The results include (1) marked displacement of the inhibitor out of the active site leading to (2) a close (2.2 A) direct contact between B (boron atom of the inhibitor) and N epsilon of His-57 and also (3) covalent bonding (1.5 A) to O gamma of Ser-195. A scheme for the mechanism of inhibition of PPE by ZAIB is proposed. A comparison with a peptidyl difluoromethyl ketone-PPE complex (Ki = 9.5 microns) is made to explain the strong inhibition of PPE by ZAIB (Ki = 0.3 micron). These results lead us to characterize this structure as a time- and space-averaged reaction intermediate, providing fresh insight into the cramped dimensions available in enzymatic catalyses.