A Molecular Basis for Innovation in Drug Excipients.

Excipients are ubiquitous in drug formulation, ensuring that active ingredient drugs are properly released on dosing, retain their properties over time, and are palatable, among other roles. Despite their crucial roles, surprisingly little is known about their systemic availability and activities on molecular targets. Here we review key excipient properties, introduce a public-accessible database that enumerates and categorizes them, and sketch a strategy for exploring their possible direct actions on molecular targets.

Structures of ceftazidime and its transition-state analogue in complex with AmpC beta-lactamase: implications for resistance mutations and inhibitor design.

Third-generation cephalosporins are widely used beta-lactam antibiotics that resist hydrolysis by beta-lactamases. Recently, mutant beta-lactamases that rapidly inactivate these drugs have emerged. To investigate why third-generation cephalosporins are relatively stable to wild-type class C beta-lactamases and how mutant enzymes might overcome this, the structures of the class C beta-lactamase AmpC in complex with the third-generation cephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determined by X-ray crystallography to 2.0 and 2.3 A resolution, respectively. Comparison of the acyl-enzyme structures of ceftazidime and loracarbef, a beta-lactam substrate, reveals that the conformation of ceftazidime in the active site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediate and the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transition state, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent for catalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-ray crystal structure of the mutant beta-lactamase GC1, which has improved activity against third-generation cephalosporins, suggests that a tandem tripeptide insertion in the Omega loop, which contains Val211, has caused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generation cephalosporins to adopt a more catalytically competent conformation. These structural differences may explain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, the complexed structure of the transition-state analogue inhibitor (K(i) 20 nM) with AmpC reveals potential opportunities for further inhibitor design.

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.

Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase.

BACKGROUND: Group I beta-lactamases are a major cause of antibiotic resistance to beta-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. RESULTS: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K(i) 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K(i) values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 A resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. CONCLUSIONS: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta-lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K(i) 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta-lactamase.

Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation.

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.

Energetic, structural, and antimicrobial analyses of beta-lactam side chain recognition by beta-lactamases.

BACKGROUND: Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these beta-lactams, most often through bacterial expression of beta-lactamases, threatens public health. To understand how beta-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because beta-lactams form covalent adducts with beta-lactamases. This has complicated functional analyses and inhibitor design. RESULTS: To investigate the contribution to interaction energy of the key amide (R1) side chain of beta-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well as four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine beta-lactamases. Therefore, binding energies can be calculated directly from K(i) values. The K(i) values measured span four orders of magnitude against the Group I beta-lactamase AmpC and three orders of magnitude against the Group II beta-lactamase TEM-1. The acylglycineboronic acids have K(i) values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of beta-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of beta-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to beta-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 A and 1.75 A resolution; these structures suggest interactions that are important to the affinity of the inhibitors. CONCLUSIONS: Acylglycineboronic acids allow us to begin to dissect interaction energies between beta-lactam side chains and beta-lactamases. Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in beta-lactam inhibitors or beta-lactam substrates of serine beta-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities. The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of anti-resistance compounds in this series.

Docking molecules by families to increase the diversity of hits in database screens: computational strategy and experimental evaluation.

Molecular docking programs screen chemical databases for novel ligands that fit protein binding sites. When one compound fits the site well, close analogs typically do the same. Therefore, many of the compounds that are found in such screens resemble one another. This reduces the variety and novelty of the compounds suggested. In an attempt to increase the diversity of docking hit lists, the Available Chemicals Directory was grouped into families of related structures. All members of every family were docked and scored, but only the best scoring molecule of a high-ranking family was allowed in the hit list. The identity and scores of the other members of these families were recorded as annotations to the best family member, but they were not independently ranked. This family-based docking method was compared with molecule-by-molecule docking in screens against the structures of thymidylate synthase, dihydrofolate reductase (DHFR), and the cavity site of the mutant T4 lysozyme Leu99 --> Ala (L99A). In each case, the diversity of the hit list increased, and more families of known ligands were found. To investigate whether the newly identified hits were sensible, we tested representative examples experimentally for binding to L99A and DHFR. Of the six compounds tested against L99A, five bound to the internal cavity. Of the seven compounds tested against DHFR, six inhibited the enzyme with apparent K(i) values between 0.26 and 100 microM. The segregation of potential ligands into families of related molecules is a simple technique to increase the diversity of candidates suggested by database screens. The general approach should be applicable to most docking methods. Proteins 2001;42:279-293.

The complexed structure and antimicrobial activity of a non-beta-lactam inhibitor of AmpC beta-lactamase.

Beta-lactamases are the major resistance mechanism to beta-lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to beta-lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non-beta-lactam inhibitors of beta-lactamases were investigated for complementarity to the structure of AmpC beta-lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo(b)thiophene-2-boronic acid (BZBTH2B), which inhibited AmpC with a Ki of 27 nM. This inhibitor is chemically dissimilar to beta-lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X-ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 A resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1-amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non-beta-lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against beta-lactams in conjunction with beta-lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many beta-lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure-based elaboration of this class of non-beta-lactam inhibitors.

Functional analyses of AmpC beta-lactamase through differential stability.

Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam adducts with Y150F were significantly destabilized relative to their WT counterparts, suggesting that this residue plays a role in recognizing the acylated intermediate of the beta-lactamase reaction. Reversible denaturation allows for energetic analyses of the complementarity of AmpC for beta-lactams, through ligand binding, and for itself, through residue substitution. Reversible denaturation may be a useful way to study ligand complementarity to other beta-lactam binding proteins as well.

Ligand solvation in molecular docking.

Solvation plays an important role in ligand-protein association and has a strong impact on comparisons of binding energies for dissimilar molecules. When databases of such molecules are screened for complementarity to receptors of known structure, as often occurs in structure-based inhibitor discovery, failure to consider ligand solvation often leads to putative ligands that are too highly charged or too large. To correct for the different charge states and sizes of the ligands, we calculated electrostatic and non-polar solvation free energies for molecules in a widely used molecular database, the Available Chemicals Directory (ACD). A modified Born equation treatment was used to calculate the electrostatic component of ligand solvation. The non-polar component of ligand solvation was calculated based on the surface area of the ligand and parameters derived from the hydration energies of apolar ligands. These solvation energies were subtracted from the ligand-receptor interaction energies. We tested the usefulness of these corrections by screening the ACD for molecules that complemented three proteins of known structure, using a molecular docking program. Correcting for ligand solvation improved the rankings of known ligands and discriminated against molecules with inappropriate charge states and sizes.

Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase.

BACKGROUND: The substrate sites of enzymes are attractive targets for structure-based inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging. RESULTS: In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and docking studies led to a derivative of one of these, dansyltyrosine (Ki 65 microM). Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 microM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes. CONCLUSIONS: TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged.

Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme.

Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30.

Structure-based design of inhibitors specific for bacterial thymidylate synthase.

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.

Three-dimensional structure of AmpC beta-lactamase from Escherichia coli bound to a transition-state analogue: possible implications for the oxyanion hypothesis and for inhibitor design.

The structures of AmpC beta-lactamase from Escherichia coli, alone and in complex with a transition-state analogue, have been determined by X-ray crystallography. The native enzyme was determined to 2.0 A resolution, and the structure with the transition-state analogue m-aminophenylboronic acid was determined to 2.3 A resolution. The structure of AmpC from E. coli resembles those previously determined for the class C enzymes from Enterobacter cloacae and Citrobacter freundii. The transition-state analogue, m-aminophenylboronic acid, makes several interactions with AmpC that were unexpected. Perhaps most surprisingly, the putative "oxyanion" of the boronic acid forms what appears to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated. Although this interaction has not previously been discussed, a carbonyl oxygen contact with the putative oxyanion or ligand carbonyl oxygen appears in most complexes involving a beta-lactam recognizing enzyme. These observations may suggest that the high-energy intermediate for amide hydrolysis by beta-lactamases and related enzymes involves a hydroxyl and not an oxyanion, although the oxyanion form certainly cannot be discounted. The involvement of the main-chain carbonyl in ligand and transition-state recognition is a distinguishing feature between serine beta-lactamases and serine proteases, to which they are often compared. AmpC may use the interaction between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabilization energy might be relieved in the transition state by a hydroxyl hydrogen bond. The structure of the m-aminophenylboronic acid adduct also suggests several ways to improve the affinity of this class of inhibitor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.

Structure-based enhancement of boronic acid-based inhibitors of AmpC beta-lactamase.

The expression of beta-lactamases is the most common form of bacterial resistance to beta-lactam antibiotics. To combat these enzymes, agents that inhibit (e.g. clavulanic acid) or evade (e.g. aztreonam) beta-lactamases have been developed. Both the beta-lactamase inhibitors and the beta-lactamase-resistant antibiotics are themselves beta-lactams, and bacteria have responded to these compounds by expressing variant enzymes resistant to inhibition (e.g. IRT-3) or that inactivate the beta-lactamase-resistant antibiotic (e.g. TEM-10). Moreover, these compounds have increased the frequency of bacteria with intrinsically resistant beta-lactamases (e.g. AmpC). In an effort to identify non-beta-lactam-based beta-lactamase inhibitors, we used the crystallographic structure of the m-aminophenylboronic acid-Escherichia coli AmpC beta-lactamase complex to suggest modifications that might enhance the affinity of boronic acid-based inhibitors for class C beta-lactamases. Several types of compounds were modeled into the AmpC binding site, and a total of 37 boronic acids were ultimately tested for beta-lactamase inhibition. The most potent of these compounds, benzo[b]thiophene-2-boronic acid (36), has an affinity for E. coli AmpC of 27 nM. The wide range of functionality represented by these compounds allows for the steric and chemical "mapping" of the AmpC active site in the region of the catalytic Ser64 residue, which may be useful in subsequent inhibitor discovery efforts. Also, the new boronic acid-based inhibitors were found to potentiate the activity of beta-lactam antibiotics, such as amoxicillin and ceftazidime, against bacteria expressing class C beta-lactamases. This suggests that boronic acid-based compounds may serve as leads for the development of therapeutic agents for the treatment of beta-lactam-resistant infections.

Flexible ligand docking using conformational ensembles.

Molecular docking algorithms suggest possible structures for molecular complexes. They are used to model biological function and to discover potential ligands. A present challenge for docking algorithms is the treatment of molecular flexibility. Here, the rigid body program, DOCK, is modified to allow it to rapidly fit multiple conformations of ligands. Conformations of a given molecule are pre-calculated in the same frame of reference, so that each conformer shares a common rigid fragment with all other conformations. The ligand conformers are then docked together, as an ensemble, into a receptor binding site. This takes advantage of the redundancy present in differing conformers of the same molecule. The algorithm was tested using three organic ligand protein systems and two protein-protein systems. Both the bound and unbound conformations of the receptors were used. The ligand ensemble method found conformations that resembled those determined in X-ray crystal structures (RMS values typically less than 1.5 A). To test the method's usefulness for inhibitor discovery, multi-compound and multi-conformer databases were screened for compounds known to bind to dihydrofolate reductase and compounds known to bind to thymidylate synthase. In both cases, known inhibitors and substrates were identified in conformations resembling those observed experimentally. The ligand ensemble method was 100-fold faster than docking a single conformation at a time and was able to screen a database of over 34 million conformations from 117,000 molecules in one to four CPU days on a workstation.

Predicting the structure of protein complexes: a step in the right direction.

In a blind test of protein-docking algorithms, six groups used different methods to predict the structure of a protein complex. All six predicted structures were close enough to the experimental complex to be useful; nevertheless, several important details of the experimental complex were missed or only partially predicted.

Molecular docking programs successfully predict the binding of a beta-lactamase inhibitory protein to TEM-1 beta-lactamase.

Crystallization of the 1:1 molecular complex between the beta-lactamase TEM-1 and the beta-lactamase inhibitory protein BLIP has provided an opportunity to put a stringent test on current protein-docking algorithms. Prior to the successful determination of the structure of the complex, nine laboratory groups were given the refined atomic coordinates of each of the native molecules. Other than the fact that BLIP is an effective inhibitor of a number of beta-lactamase enzymes (KI for TEM-1 approximately 100 pM) no other biochemical or structural data were available to assist the practitioners in their molecular docking. In addition, it was not known whether the molecules underwent conformational changes upon association or whether the inhibition was competitive or non-competitive. All six of the groups that accepted the challenge correctly predicted the general mode of association of BLIP and TEM-1.

Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive.

A number of mutations have been shown previously to stabilize T4 lysozyme. By combining up to seven such mutations in the same protein, the melting temperature was incrementally increased by up to 8.3 degrees C at pH 5.4 (delta delta G = 3.6 kcal/mol). This shows that it is possible to engineer a protein of enhanced thermostability by combining a series of rationally designed point mutations. It is also shown that this stabilization is achieved with only minor, localized changes in the structure of the protein. This is consistent with the observation that the change in stability of each of the multiple mutants is, in each case, additive, i.e. equal to the sum of the stability changes associated with the constituent single mutants. One of the seven substitutions, Asn116-->Asp, changes a residue that participates in substrate binding; not surprisingly, it causes a significant loss in activity. Ignoring this mutation, there is a gradual reduction in activity as successively more mutations are combined.

A relationship between protein stability and protein function.

Enzymes are thought to use their ordered structures to facilitate catalysis. A corollary of this theory suggests that enzyme residues involved in function are not optimized for stability. We tested this hypothesis by mutating functionally important residues in the active site of T4 lysozyme. Six mutations at two catalytic residues, Glu-11 and Asp-20, abolished or reduced enzymatic activity but increased thermal stability by 0.7-1.7 kcal.mol-1. Nine mutations at two substrate-binding residues, Ser-117 and Asn-132, increased stability by 1.2-2.0 kcal.mol-1, again at the cost of reduced activity. X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme. In two of these structures the enzyme undergoes a general conformational change, similar to that seen in an enzyme-product complex. These results support a relationship between stability and function for T4 lysozyme. Other evidence suggests that the relationship is general.