Search

Pipecolic acid derivatives have proven to be effective P′1 groups in a series of highly potent inhibitors of the enzyme HIV protease. One such inhibitor, Ro 31-8959, contains the saturated bicyclic ring system decahydroisoquinoline (DIQ) in the P′1 position. The binding orientation of Ro 31-8959 is known from X-ray crystallography. However, the bound conformation of the S-hydroxy diastereomer has not been studied, and for this molecule there are at least two different possible binding conformations. Specifically, the N-alkyl substituents may be equatorial or axial and the 3-carboxamide may be rotated into several different orientations. To gain a better understanding of the relative energies of these various conformations, ab initio molecular orbital calculations have been carried out on a series of pipecolic acid and DIQ derivatives. The results indicate that the lowest-energy N-equatorial rotamer is always at least 3 kcal/mol more stable than the lowest-energy N-axial rotamer. The presence of the second ring, as in the DIQ system, considerably raises the equatorial–axial difference to nearly 7 kcal/mol. Also, the preferred rotation angle of the amide group is different for the N-equatorial and N-axial cases. When the molecular dynamics-averaged conformation of the bound S-hydroxy inhibitor is considered, the energy difference between the N-equatorial and N-axial conformers drops to 4–5 kcal/mol. The preferred amide rotations in these systems are compared to those found in proline-containing peptides. Finally, some observations are made with respect to the large conformational energy penalty necessary for binding Ro 31-8959. © John Wiley & Sons, Inc.

We describe a new method for de novo design of molecules that bind to protein active sites. The method, CONCEPTS (Creation of Novel Compounds by Evaluation of Particles at Target Sites), places a group of atom-like particles in the site. The particles are free to move within the site to improve binding to the protein. A key innovation of this technique is that covalent connections are made among the particles in a stochastic and dynamically reversible manner. These changes in the topology are either accepted or rejected depending on their ability to improve the total energy of the enzyme–inhibitor complex. The method is applied to two test systems: The FK506 binding protein (FKBP-12) and HIV-1 aspartyl protease. In both cases, we are able to predict, de novo, drugs that have striking similarities to known potent inhibitors and that can successfully be used to generate “hits” of the known inhibitors from a data base. © John Wiley & Sons, Inc.

A novel method for de novo drug design, GroupBuild, has been developed to suggest chemically reasonable structures which fill the active sites of enzymes. The proposed molecules provide good steric and electrostatic contact with the enzyme and exist in low-energy conformations. These structures are composed entirely of individual functional groups (also known as "building blocks" or "fragments") which the program chooses from a predefined library. User-selected enzyme seed atom(s) may be used to determine the area(s) in which structure generation begins. Alternatively, GroupBuild may begin with a predocked "inhibitor core" from which fragments are grown. For each new fragment generated by the program, several thousand candidates in a variety of locations and orientations are considered. Each of these candidates is scored based on a standard molecular mechanics potential function. The selected fragment and orientation are chosen from among the highest scoring cases. Tests of the method using HIV protease, FK506 binding protein, and human carbonic anhydrase demonstrate that structures similar to known potent inhibitors may be generated with GroupBuild.

Ab initio calcutions have been used to study the changes in energy of ethylene glycol and 1,2-dimethoxyethane as a function of rotation around the central C-C bond. Geometries have been fully optimized at the 3-21G and 6-31G * levels, and single-point calculations have been carried out at higher levels (up to 6-311++G ** for ethylene glycol and 6-31+G * for 1,2-dimethoxyethane), including electron correlation up to MP4(SDTQ). For ethylene glycol, the H-O-C-C angles were started in a trans orientation to prevent intramolecular hydrogen bonding

The mechanism of FK506 immunosuppression has been proposed to proceed by formation of a tight-binding complex with the intracellular 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex then acts as a specific high-affinity inhibitor of the intracellular protein phosphatase PP2B (calcineurin), interrupting downstream dephosphorylation events required for T-cell activation. Site-directed mutagenesis of many of the surface residues of FKBP12 has no significant effect on its affinity for calcineurin. We have identified, however, three FKBP12 surface residues (Asp-37, Arg-42, and His-87) proximal to a solvent-exposed segment of bound FK506 that may be direct contacts in the calcineurin complex. Site-directed mutagenesis of two of these residues decreases the affinity of FKBP12-FK506 for calcineurin (Ki) from 6 nM for wild-type FKBP12 to 3.7 microM for a R42K/H87V double mutant, without affecting the peptidylprolyl isomerase activity or FK506 affinity of the mutant protein. These FKBP12 mutations along with several substitutions on FK506 known to affect calcineurin binding form a roughly 100-A2 region of the FKBP12-FK506 complex surface that is likely to be within the calcineurin binding site.

Modern drug design increasingly relies on structural information for the improvement of in vitro potency. Recently, these methods have been used to optimize in vitro properties as well. Barries to the sucessful integration of these technologies within the drug industry seem to be largely cultural rather than scientific.

A FKB cDNA encoding murine FK506 binding protein (FKBP) has been cloned, and its complete nucleotide sequence has been determined. The open reading frame within the 1556-bp cDNA segment encodes an 108 amino acid (aa) protein that differs from the human FKBP by three aa and from the bovine FKBP by five aa. Molecular modeling of the protein places the aa substitutions at positions not directly involved in drug binding or interaction with the potential drug target protein, calcineurin A.

A series of 5-substituted thieno[2,3-b]- and thieno[3,2-b)- and thieno[3,2-b)thiophene-2-sulfonamides was prepared and evaluated for topical ocular hypotensive activity in glaucoma models. The 5-substituents were varied to maximize both inhibitory potency against carbonic anhydrase and water solubility. At the same time, these substituents were varied in order to obtain compounds with the appropriate pKa to minimize pigment binding in the iris. All of these variables were optimized in the best compound, 5-[[(methoxyethyl)[(methoxyethyl)ethyl] amino]methyl]thieno[2,3-b]thiophene-2-sulfonamide hydrochloride (55).

1,2-Disubstituted ethanes having strongly electronegative substituents prefer the gauche conformation over the usual trans form. This is not the result of an attractive interaction in the gauche form, but rather a destabilizing interaction in the trans rotamer. The interaction appears to be the result of the formation of bent bonds, and the lower stability of a twist bent bond over that of syn bent bond. The lower energy of cis-1,2-difluoroethene as compared to its trans form may be explained in the same way.

Small molecule protein kinase inhibitors are widely employed as biological reagents and as leads in the design of drugs for a variety of diseases. One of the hardest challenges in kinase inhibitor design is achieving target selectivity. By utilizing X-ray structural information for four promiscuous inhibitors, we propose a five-point pharmacophore for kinase frequent hitters, demonstrate its ability to discriminate between frequent hitters and selective ligands, and suggest a strategy for selective inhibitor design.

Recent developments in combinatorial chemistry and high-throughput screening have dramatically increased the scale on which drug discovery programs are carried out. Along with these advances has come a need for automated methods of determining which compounds from a library should be synthesized and screened. These methods range from simple counting schemes to sophisticated machine learning techniques such as neural networks. While many of these methods have performed well in validation studies, the field is still in its formative stage. This paper reviews a number of computational techniques for identifying drug-like molecules and examines challenges facing the field.

We continue our study of the common features present in drug molecules by looking in detail at drug side chains. Using shape description methods, we divide a database of commercially available drugs into a list of common drug side chains. On the basis of the atom pair shape descriptor (taking into account atom type, hybridization, and bond order), there are 1,246 different side chains among the 5,090 compounds analyzed. The average number of side chains per molecule is 4, and the average number of heavy atoms per side chain is 2. If we ignore the carbonyl side chain, then there are approximately 15,000 occurrences of side chains. Of these 15,000 approximately 11,000 are from the "top 20" group of side chains. This suggests that the diversity that side chains provide to drug molecules is quite low. We discuss ways that this work could be used to provide guidance for molecular design efforts.

We present the results of an extensive computational study in which we show that combining scoring functions in an intersection-based consensus approach results in an enhancement in the ability to discriminate between active and inactive enzyme inhibitors. This is illustrated in the context of docking collections of three-dimensional structures into three different enzymes of pharmaceutical interest: p38 MAP kinase, inosine monophosphate dehydrogenase, and HIV protease. An analysis of two different docking methods and thirteen scoring functions provides insights into which functions perform well, both singly and in combination. Our data shows that consensus scoring further provides a dramatic reduction in the number of false positives identified by individual scoring functions, thus leading to a significant enhancement in hit-rates.

Publisher Summary This chapter focuses on structure-based drug design, which is a general term for using protein structural information from X-ray crystallography or NMR spectroscopy to assist in the design of novel therapeutic agents. Structure-based drug design is currently most applicable to developing inhibitors that bind to enzymes. Overall, approximately one-third of marketed drugs function primarily through direct inhibition of enzyme activity. Over 200 enzymes have been proposed as therapeutic targets within the pharmaceutical industry. New enzyme inhibitors have great potential to become therapeutically useful in the clinic and successful in the market. Detailed analysis of drug targets indicates that they cover a very broad spectrum of enzyme activities as well as being structurally diverse. This implies that efficient enzyme inhibitor design requires flexibility in the implementation of enzymatic assays as well as an appreciation of the subtleties of protein structure. The chapter reviews recent examples of the design of drugs that inhibit enzymes of therapeutic interest. An overview of structural genomics and structure-based drug design in presented and diverse structures and functions of therapeutically relevant targets are also explained.

Background: The p38 mitogen-activated protein (MAP) kinase regulates signal transduction in response to environmental stress. Pyridinylimidazole compounds are specific inhibitors of p38 MAP kinase that block the production of the cytokines interleukin-1 β and tumor necrosis factor α, and they are effective in animal models of arthritis, bone resorption and endotoxin shock. These compounds have been useful probes for studying the physiological functions of the p38-mediated MAP kinase pathway. Results: We report the crystal structure of a novel pyridinylimidazole compound complexed with p38 MAP kinase, and we demonstrate that this compound binds to the same site on the kinase as does ATP. Mutagenesis showed that a single residue difference between p38 MAP kinase and other MAP kinases is sufficient to confer selectivity among pyridinylimidazole compounds. Conclusions: Our results reveal how pyridinylimidazole compounds are potent and selective inhibitors of p38 MAP kinase but not other MAP kinases. It should now be possible to design other specific inhibitors of activated p38 MAP kinase using the structure of the nonphosphorylated enzyme.

An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 A resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a β sheet of the enzyme core.

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolyl isomerase that binds the macrolides FK506 and rapamycin. We have examined the role of the binding pocket residues of FKBP12 in protein-ligand interactions by making conservative substitutions of 12 of these residues by site-directed mutagenesis. For each mutant FKBP12, we measured the affinity for FK506 and rapamycin and the catalytic efficiency in the cis-frans peptidyl-prolyl isomerase reaction. The mutation of Trp59 or Phe99 generates an FKBP12 with a significantly lower affinity for FK506 than wild-type protein. Tyr26 and Tyr82 mutants are enzymatically active, demonstrating that hydrogen bonding by these residues is not required for catalysis of the cis-trans peptidyl-prolyl isomerase reaction, although these mutations alter the substrate specificity of the enzyme. We conclude that hydrophobic interactions in the active site dominate in the stabilization of FKBP12 binding to macrolide ligands and to the twisted-amide peptidyl-prolyl substrate intermediate.

VX-478 belongs to a novel class of HIV-1 protease inhibitors that are based on N,N-disubstituted benzene sulfonamides. Force field parameters for the N,N-dialkyl benzene sulfonamide moiety have been assembled from the literature and from our own ab initio calculations. These parameters were employed to calculate solvation and binding free energy differences between VX-478 and two analogs. The free energy perturbation method has been used to determine these differences using two approaches. In the first approach, intergroup interaction terms only were included in the calculation of free energies (as in most reports of free energy calculations using AMBER). In the second approach, both the inter- and intragroup interaction terms were included. The results obtained with the two approaches are in excellent agreement with each other and are also in close agreement with the experimental results. The solvation free energies of N,N-dimethyl benzene sulfonamide derivatives (truncated models of the inhibitors), calculated using continuum solvation (AMSOL) methods, are found to be in qualitative agreement with the experimental and free energy perturbation results. The binding and solvation free energy results are discussed in the context of structure-based drug design to show how physicochemical properties (for example aqueous solubilities and bioavailabilities) of these HIV-1 protease inhibitors were improved, while maintaining their inhibitory potency.

We have synthesized a series of non-macrocyclic ligands to FKBP12 that are comparable in binding potency and peptidyl prolyl isomerase (PPIase) inhibition to FK506 itself. We have also solved the structure of one of these ligands in complex with FKBP12, and have compared that structure to the FK506–FKBP12 complex. Consistent with the observed inhibitory equipotency of these compounds, we observe a strong similarity in the conformation of the two ligands in the region of the protein that mediates PPIase activity. Our compounds, however, are not immunosuppressive. In the FKBP12–FK506 complex, a significant portion of the FK506 ligand, its `effector domain', projects beyond the envelope of the binding protein in a manner that is suggestive of a potential interaction with a second protein, the calcium-dependent phosphatase, calcineurin, whose inhibition by the FKBP 12–FK506 complex interrupts the T-cell activation events leading to immunosuppression. In contrast, our compounds bind within the surface envelope of FKBP12, and induce significant changes in the structure of the FKBP12 protein which may also affect calcineurin binding indirectly.

DNA damaging agents have been the cornerstone of solid cancer therapy for decades yet they provide only modest benefit for patients with many tumor types. This reflects, in part, the efficient repair of DNA damage via a complex signaling and repair network known as the DNA damage response (DDR). Key regulators of the DDR are the phosphoinositol 3-kinase-like serine/threonine protein kinase (PIKK) family members ATR, ATM and DNA-PK. The DDR acts to detect DNA lesions, enforce checkpoints to halt cell cycle progression, and stimulate repair. Recent data have shown that elements of the DDR are commonly defective in cancer cells. It is widely believed that these cells become dependent on the remaining DDR pathways for survival from DNA damage. Inhibitors have been reported for a number of DDR enzymes, including ATM, DNA-PK, CHK1 and PARP, however there are no reports of drug-like ATR inhibitors. Here we disclose the in vitro characterization of a potent and highly selective ATR inhibitor (VE-821). This compound selectively blocks ATR signaling in cells (IC50 = 0.7 µM), but has little impact on ATM or DNA-PK signaling (IC50 >10 µM). Treatment with 10 µM VE-821 for 144 h causes little cell death in normal cell lines (5-11 %) but markedly higher death in cancer cell lines (28-46 %). VE-821 also dramatically sensitizes many cancer cells to multiple classes of genotoxic agents including antimetabolites, topoisomerase inhibitors and crosslinking agents; with over 10-fold increases genotoxic potency observed in some cases. In a panel of 36 lung cancer cell lines, VE-821 sensitized the cytotoxic effect of cisplatin to a far greater magnitude and over a broader subset of these lines than potent inhibitors of ATM, Chk1, or PARP. In over half of these cell lines, the IC50 of cisplatin was reduced by greater than 5 fold upon the addition of VE-821. We show that a basis for the cancer-selective effects of VE-821 is a synthetic lethal interaction between loss of ATM signaling (a frequent event in cancer resulting from loss of function of proteins such as ATM or p53) and ATR inhibition when cells encounter DNA damage. In keeping with this, ATR inhibition does not sensitize normal cells (with functional ATM) to the cytotoxic effects of genotoxic therapy. In this case a compensatory DDR is activated that is associated with marked activation of ATM, which in turn leads to reversible checkpoint arrest and a strong survival response. These studies show for the first time that a selective ATR inhibitor can preferentially sensitize cancer cells to genotoxic drugs by exploiting a synthetic lethal interaction between ATM and ATR signaling. This underpins the broad potential of ATR inhibition as a highly promising new strategy to improve the efficacy of genotoxic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2011-5491

A novel method, which we call GenStar, has been developed to suggest chemically reasonable structures which fill the active sites of enzymes. The proposed molecules provide good steric contact with the enzyme and exist in low-energy conformations. These structures are composed entirely of sp3 carbons which are grown sequentially, but which can also branch or form rings. User-selected enzyme seed atoms may be used to determine the area in which structure generation begins. Alternatively, GenStar may begin with a predocked 'inhibitor core' from which atoms are grown. For each new atom generated by the program, several hundred candidate positions representing a range of reasonable bond lengths, bond angles, and torsion angles are considered. Each of these candidates is scored, based on a simple enzyme contact model. The selected position is chosen at random from among the highest scoring cases. Duplicate structures may be removed using a variety of criteria. The compounds may be energy minimized and displayed using standard modeling programs. Also, it is possible to analyze the collection of all structures created by GenStar and locate binding motifs for common fragments such as benzene and naphthylene. Tests of the method using HIV protease, FK506 binding protein (FKBP-12) and human carbonic anhydrase (HCA-II) demonstrated that structures similar to known potent inhibitors may be generated with GenStar.

The properties of a molecule are determined by the distribution of its electrons. This distribution can be described by the charge density, which is readily obtained from the wave functions derived by ab initio molecular orbital calculations. The charge density may be analyzed in a number of different fashions to give information about the effects of substituents, structural changes, and electronic excitation on the properties of molecules; one common procedure makes use of projection density or charge difference plots. Charge density also may be partitioned among atoms, and by numerical integration over appropriate volume elements one may obtain atomic charges, dipoles, kinetic energies, and other properties of the atoms in a molecule. Many chemical phenomena have been analyzed in terms of charge densities.