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Several efficient correspondence graph-based algorithms for determining the maximum common substructure (MCS) of a pair of molecules have been published in the literature. The extension of the problem to three or more molecules is however nontrivial; heuristics used to increase the efficiency in the two-molecule case are either inapplicable to the many-molecule case or do not provide significant speedups. Our specific algorithmic contribution is two-fold. First, we show how the correspondence graph approach for the two-molecule case can be generalized to obtain an algorithm that is guaranteed to find the optimum connected MCS of multiple molecules, and that runs fast on most families of molecules using a new divide-and-conquer strategy that has hitherto not been reported in this context. Second, we provide a characterization of those compound families for which the algorithm might run slowly, along with a heuristic for speeding up computations on these families. We also extend the above algorithm to a heuristic algorithm to find the disconnected MCS of multiple molecules and to an algorithm for clustering molecules into groups, with each group sharing a substantial MCS. Our methods are flexible in that they provide exquisite control on various matching criteria used to define a common substructure.

The Protein Data Bank is the most comprehensive source of experimental macromolecular structures. It can, however, be difficult at times to locate relevant structures with the Protein Data Bank search interface. This is particularly true when searching for complexes containing specific interactions between protein and ligand atoms. Moreover, searching within a family of proteins can be tedious. For example, one cannot search for some conserved residue as residue numbers vary across structures. We describe herein three databases, Protein Relational Database, Kinase Knowledge Base, and Matrix Metalloproteinase Knowledge Base, containing protein structures from the Protein Data Bank. In Protein Relational Database, atom-atom distances between protein and ligand have been precalculated allowing for millisecond retrieval based on atom identity and distance constraints. Ring centroids, centroid-centroid and centroid-atom distances and angles have also been included permitting queries for pi-stacking interactions and other structural motifs involving rings. Other geometric features can be searched through the inclusion of residue pair and triplet distances. In Kinase Knowledge Base and Matrix Metalloproteinase Knowledge Base, the catalytic domains have been aligned into common residue numbering schemes. Thus, by searching across Protein Relational Database and Kinase Knowledge Base, one can easily retrieve structures wherein, for example, a ligand of interest is making contact with the gatekeeper residue.

In this paper, we describe a combination of structural informatics approaches developed to mine data extracted from existing structure knowledge bases (Protein Data Bank and the GVK database) with a focus on kinase ATP-binding site data. In contrast to existing systems that retrieve and analyze protein structures, our techniques are centered on a database of ligand-bound geometries in relation to residues lining the binding site and transparent access to ligand-based SAR data. We illustrate the systems in the context of the Abelson kinase and related inhibitor structures., (2009 Elsevier Ltd. All rights reserved.)

A novel algorithm for the connecting of fragment molecules is presented and validated for a number of test systems. Within the CONFIRM (Connecting Fragments Found in Receptor Molecules) approach a pre-prepared library of bridges is searched to extract those which match a search criterion derived from known experimental or computational binding information about fragment molecules within a target binding site. The resulting bridge 'hits' are then connected, in an automated fashion, to the fragments and docked into the target receptor. Docking poses are assessed in terms of root-mean-squared deviation from the known positions of the fragment molecules, as well as docking score should known inhibitors be available. The creation of the bridge library, the full details and novelty of the CONFIRM algorithm, and the general applicability of this approach within the field of fragment-based de novo drug design are discussed.

The cyclin-dependent kinases (CDKs), as complexes with their respective partners, the cyclins, are critical regulators of cell cycle progression. Because aberrant regulations of CDK4/cyclin D1 lead to uncontrolled cell proliferation, a hallmark of cancer, small-molecule inhibitors of CDK4/cyclin D1 are attractive as prospective antitumor agents. The series of 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione derivatives reported here represents a novel class of potent inhibitors that selectively inhibit CDK4 over CDK2 and CDK1 activities. In the headpiece of the 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione, a basic amine substituent is required on the aniline ring for the CDK4 inhibitory activity. The inhibitory activity is further enhanced when an aryl or heteroaryl substituent is introduced at the C-6 position of the isoquinoline-1,3(2H,4H)-dione core. We present here SAR data and a CDK4 mimic model that explains the binding, potency, and selectivity of our CDK4 selective inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

ZipA is a membrane anchored protein in Escherichia coli that interacts with FtsZ, a homolog of eukaryotic tubulins, forming a septal ring structure that mediates bacterial cell division. Thus, the ZipA/FtsZ protein-protein interaction is a potential target for an antibacterial agent. We report here an NMR-based fragment screening approach which identified several hits that bind to the C-terminal region of ZipA. The screen was performed by 1H-15N HSQC experiments on a library of 825 fragments that are small, lead-like, and highly soluble. Seven hits were identified, and the binding mode of the best one was revealed in the X-ray crystal structure. Similar to the ZipA/FtsZ contacts, the driving force in the binding of the small molecule ligands to ZipA is achieved through hydrophobic interactions. Analogs of this hit were also evaluated by NMR and X-ray crystal structures of these analogs with ZipA were obtained, providing structural information to help guide the medicinal chemistry efforts.

In earlier work from our laboratory, we have described the use of the ring system and ring scaffold as descriptors. We showed that these descriptors could be used for fast compound clustering, novelty determination, compound acquisition, and combinatorial library design. Here we extend the concept to a whole family of structural descriptors with the ring system as the centerpiece. We show how this simple idea can be used to build powerful search tools for mining chemical databases in useful ways. We have also built recursive partition trees using these fragments as descriptors. We will discuss how these trees can help in analyzing complex structure-activity data.

HER-2 belongs to the ErbB family of receptor tyrosine kinases, which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25-30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Trastuzumab (Herceptin), a monoclonal antibody to HER-2, is specifically approved for HER-2-positive breast cancer but is active only in a subset of these tumors. Blocking HER-2 function by a small molecule kinase inhibitor, therefore, represents an attractive alternate strategy to inhibit the growth of HER-2-positive tumors. HKI-272 is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. HKI-272 reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor, most likely by targeting a cysteine residue in the ATP-binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, HKI-272 treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. This leads to arrest at the G(1)-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle, ultimately resulting in decreased cell proliferation. In vivo, HKI-272 is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, HKI-272 has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers.

The syntheses and EGFR kinase inhibitory activity of a series of 6-substituted-4-anilino [1,7] and [1,8] naphthyridine-3-carbonitriles are described. Both reversible and irreversible binding inhibitors were prepared. These series were compared with each other and with the corresponding 4-anilinoquinoline-3-carbonitriles. Compounds having a 1,7-naphthyridine core structure can retain high potency while those with a 1,8-naphthyridine core are significantly less active. These results are consistent with molecular modeling observations.

2,3-Dihydro-1,4-benzodioxanes with aryl 8-aza-bicyclo[3.2.1]oct-3-ene attachments 2 produce compounds with potent 5-HT-T affinity, and weak 5-HT(1A) affinity and alpha(1) affinity. This compares with 2,3-dihydro-1,4-benzodioxanes containing 8-aza-bicyclo[3.2.1] octan-3-ol attachments 4 which possess potent 5-HT(1A) affinity, moderate to good selectivity over alpha(1) and little 5-HT-T affinity. A 3-benzothiophene analogue of 4 (30) was synthesized which possesses potent 5-HT(1A) affinity and especially good selectivity over both alpha(1) and 5-HT-T.

Most current methods for the design of pharmaceutical screening libraries centre around compound diversity. We present arguments for a different approach, involving a fixed number of analogs around a set of medicinally relevant scaffolds. Most current approaches to screening library design emphasize wide coverage of chemical space at the expense of poor local representation. We propose constructing uniform libraries around fixed ring scaffolds with "adequate" representation so as not to miss potentially active series.

A series of of 6,7-disubstituted-4-anilinoquinoline-3-carbonitrile derivatives that function as irreversible inhibitors of EGFR and HER-2 kinases have been prepared. These inhibitors have, at the 6-position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solublilizing substituents. These compounds were prepared by acylation of 6-amino-4-(arylamino)quinoline-3-carbonitriles with unsaturated acid chlorides or mixed anhydrides. We performed competitive reactivity studies showing that attaching a dialkylamino group onto the end of the Michael acceptor results in compounds with greater reactivity due to intramolecular catalysis of the Michael addition. This, along with improved water-solubility results in compounds with enhanced biological properties. We present molecular modeling results consistent with the proposed mechanism of inhibition. One compound, 5 (EKB-569), which shows excellent oral in vivo activity, was selected for further studies and is currently in phase I clinical trials for the treatment of cancer.

The syntheses and biological evaluations of 4-anilinoquinoline-3-carbonitrile analogues of the three clinical lead 4-anilinoquinazolines Iressa, Tarceva, and CI-1033 are described. The EGFR and HER-2 kinase inhibitory activities and the cell growth inhibition of the two series are compared with each other and with the clinical lead EKB-569. Similar activities are observed between these two series.

We address the problem of designing a general-purpose combinatorial library to screen for pharmaceutical leads. Conventional approaches focus on diversity as the primary factor in designing such libraries. We suggest making screening libraries out of a set of pharmaceutically relevant scaffolds, with multiple analogs per scaffold. The rationale for this rests on the fact that even though the hit-rate in active series is much higher than in the database as a whole, often a large fraction of the compounds in active series are inactive. This is especially true when the series has not been optimized for the target under study. We introduce the concept of hit-rate within a series and use historic screening data to arrive at a crude estimate for it. We then use simple probability arguments to show that 50-100 compounds are required in each series in order to be nearly certain of finding at least one active compound in each true active series for any given target.

It has been shown previously that 4-anilino quinazolines compete with the ability of ATP to bind the epidermal growth factor receptor (EGF-R), inhibit EGF-stimulated autophosphorylation of tyrosine residues in EGF-R, and block EGF-mediated growth. Since millimolar concentrations of ATP in cells could reduce the efficacy of 4-anilino quinazolines in cells and the activity of these compounds would not be sustained once they were removed from the body, we reasoned that irreversible inhibitors of EGF-R might improve the activity of this series of compounds in animals. Molecular modeling of the EGF-R kinase domain was used to design irreversible inhibitors. We herein describe one such inhibitor: N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide, known as CL-387,785. This compound covalently bound to EGF-R. It also specifically inhibited kinase activity of the protein (IC50 = 370+/-120 pM), blocked EGF-stimulated autophosphorylation of the receptor in cells (ic50 approximately 5 nM), inhibited cell proliferation (IC50 = 31-125 nM) primarily in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2, and profoundly blocked the growth of a tumor that overexpresses EGF-R in nude mice (when given orally at 80 mg/kg/day for 10 days, daily). We conclude that CL-387,785 is useful for studying the interaction of small molecules with EGF-R and may have clinical utility.

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.

We present some new ideas for characterizing and comparing large chemical databases. The comparison of the contents of large databases is not trivial since it implies pairwise comparison of hundreds of thousands of compounds. We have developed methods for categorizing compounds into groups or series based on their ring-system content, using precalculated structure-based hashcodes. Two large databases can then be compared by simply comparing their hashcode tables. Furthermore, the number of distinct ring-system combinations can be used as an indicator of database diversity. We also present an independent technique for diversity assessment called the saturation diversity approach. This method is based on picking as many mutually dissimilar compounds as possible from a database or a subset thereof. We show that both methods yield similar results. Since the two methods measure very different properties, this probably says more about the properties of the databases studied than about the methods.

A novel method for generation of chemical structures of potential pharmaceutical interest is presented. Structures are generated by random combination of known fragments and selected by statistical topological techniques. The power of the method lies in the great profusion of candidates generated together with the extremely high selectivity imposed by the techniques of selection.

We develop an extension of conventional distance geometry techniques that treats two or more molecules as a single "ensemble". This extension can be used to find a common pharmacophore, i.e., the spatial arrangement of essential groups, from a small set of biologically active molecules. The approach can generate, in one step, coordinates for the set of molecules in their "active" conformations such that their essential groups are superimposed. As an example, we show how the nicotinic pharmacophore can be deduced from a set of four nicotinic agonists: nicotine, cytisine, ferruginine methiodide, and muscarone. Three essential groups in each agonist are chosen: the cationic center (A), an electronegative atom (B), and an atom (C) that forms a dipole with B. There is only one pharmacophore possible for the superposition of these essential groups: a triangle with sides 4.8 A (A-B), 4.0 A (A-C), and 1.2 A (B-C). The pharmacophore triangle, which is consistent with previous models in the literature, can also be achieved by the agonist trans-3,3'-bis[(trimethylammonio)methyl]azobenzene and the antagonists strychnine, trimethaphan, and dihydro-beta-erythroidine. An examination of the common volumes of agonists suggests a specific disposition of molecular volume relative to the pharmacophore triangle. We discuss the relative strengths and drawbacks of the ensemble approach vs. other conformational search methods.

Calicheamicin gamma 1I is a recently discovered diyne-ene-containing antitumor antibiotic that cleaves DNA in a double-stranded fashion, a rarity among drugs, at specific sequences. It is proposed that the cutting specificity is due to a combination of the complementarity of the diyne-ene portion of the aglycone with DNA secondary structures and stabilization by association of the thiobenzoate-carbohydrate tail with the minor groove.