Application and utilization of chemoinformatics tools in lead generation and optimization.

The process of Drug Discovery is a complex and high risk endeavor that requires focused attention on experimental hypotheses, the application of diverse sets of technologies and data to facilitate high quality decision-making. All is aimed at enhancing the quality of the chemical development candidate(s) through clinical evaluation and into the market. In support of the lead generation and optimization phases of this endeavor, high throughput technologies such as combinatorial/high throughput synthesis and high throughput and ultra-high throughput screening, have allowed the rapid analysis and generation of large number of compounds and data. Today, for every analog synthesized 100 or more data points can be collected and captured in various centralized databases. The analysis of thousands of compounds can very quickly become a daunting task. In this article we present the process we have developed for both analyzing and prioritizing large sets of data starting from diversity and focused uHTS in support of lead generation and secondary screens supporting lead optimization. We will describe how we use informatics and computational chemistry to focus our efforts on asking relevant questions about the desired attributes of a specific library, and subsequently in guiding the generation of more information-rich sets of analogs in support of both processes.

Library design practices for success in lead generation with small molecule libraries.

The generation of novel structures amenable to rapid and efficient lead optimization comprises an emerging strategy for success in modern drug discovery. Small molecule libraries of sufficient size and diversity to increase the chances of discovery of novel structures make the high throughput synthesis approach the method of choice for lead generation. Despite an industry trend for smaller, more focused libraries, the need to generate novel lead structures makes larger libraries a necessary strategy. For libraries of a several thousand or more members, solid phase synthesis approaches are the most suitable. While the technology and chemistry necessary for small molecule library synthesis continue to advance, success in lead generation requires rigorous consideration in the library design process to ensure the synthesis of molecules possessing the proper characteristics for subsequent lead optimization. Without proper selection of library templates and building blocks, solid phase synthesis methods often generate molecules which are too heavy, too lipophilic and too complex to be useful for lead optimization. The appropriate filtering of virtual library designs with multiple computational tools allows the generation of information-rich libraries within a drug-like molecular property space. An understanding of the hit-to-lead process provides a practical guide to molecular design characteristics. Examples of leads generated from library approaches also provide a benchmarking of successes as well as aspects for continued development of library design practices.

Stereochemical control factors in the Hantzsch thiazole synthesis: a Hammett substitution correlation analysis.

[reaction--see text] It is possible to correlate the distribution of stereochemical products produced during a Hantzsch thiazole synthesis according to the Hammett free-energy equation. This analysis confirms the presumed control of the rate of epimerization during thiazole formation due to stabilization of a cationic transition state intermediate during dehydration of the thiazoline ring system. In the chemical system under study, the stereochemical outcome of the reaction also appears to occur according to a kinetically controlled protonation of a thiazoline tautomer.

Current practices in generation of small molecule new leads.

The current drug discovery processes in many pharmaceutical companies require large and growing collections of high quality lead structures for use in high throughput screening assays. Collections of small molecules with diverse structures and "drug-like" properties have, in the past, been acquired by several means: by archive of previous internal lead optimization efforts, by purchase from compound vendors, and by union of separate collections following company mergers. More recently, many drug discovery companies have established dedicated efforts to effect synthesis by internal and/or outsourcing efforts of targeted compound libraries for new lead generation. Although high throughput/combinatorial chemistry is an important component in the process of new lead generation, the selection of library designs for synthesis and the subsequent design of library members has evolved to a new level of challenge and importance. The potential benefits of screening multiple small molecule compound library designs against multiple biological targets offers substantial opportunity to discover new lead structures. Subsequent optimization of such compounds is often accelerated because of the structure-activity relationship (SAR) information encoded in these lead generation libraries. Lead optimization is often facilitated due to the ready applicability of high-throughput chemistry (HTC) methods for follow-up synthesis. Some of the strategies, trends, and critical issues central to the success of lead generation processes are discussed below.

The sugars in chromomycin A3 stabilize the Mg(2+)-dimer complex.

Chromomycin A3 (CRA3) is a glycosylated antitumor antibiotic that binds as a dimer to the minor groove of DNA, with a Mg2+ cation (or another divalent cation with a radius less than 0.85 A) forming the center of the dimer. It has been shown that the chromose sugars are necessary for DNA binding [Kaziro & Kamiyama (1967) J. Biochem. (Tokyo) 62, 424-429; Kamiyama (1968) J. Biochem. (Tokyo) 63, 566-572], although the reason for this has not been addressed. We have investigated the role that the chromose sugars play in metal complexation in solution (methanol) by comparing the optical behavior of CRA3 and its aglycon, CRN, in the presence of various divalent metals (Mg2+, Ni2+, and Ca2+). The results show that CRA3 forms a dimeric complex [i.e., (CRA3)2M, where M is a metal ion] in the presence of 1 mol equiv of either Ni2+ or Mg2+ but a 1:1 complex in the presence of the much larger Ca2+. In contrast, CRN forms a 1:1 complex (CRN.M)+ with all three metals under identical conditions (1:1 mole ratio of drug to metal). Thus, for the smaller metal ions the sugars stabilize the 2:1 CRA3-metal complex in solution. NMR data on the 2:1 CRA3-Mg2+ complex show that the trisaccharide of one CRA3 molecule lies in close proximity to the chromophore of the other CRA3 molecule. This interaction, which is also present in the Mg(2+)-CRA3-DNA complex [Gao & Patel (1989) Biochemistry 28, 751-762], appears to be related to the stability of the dimer in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Quisqualate-sensitive glutamate receptors of the locust Schistocerca gregaria are antagonised by intracellularly applied philanthotoxin and spermine.

The effects of intracellularly and extracellularly applied synthetic analogues of delta-philanthotoxin (PhTX-433) and the polyamine spermine on the excitatory postsynaptic current (EPSC) of glutamatergic synapses and single channel currents gated by quisqualate-sensitive glutamate receptors (QUIS-R) on locust leg muscle have been compared. When applied extracellularly all 3 compounds reversibly antagonised the EPSC and the single channel currents. Antagonism was voltage independent, but use (agonist) dependent. Antagonism also occurred when they were injected into muscle fibres, but in this case it was not use dependent. It is proposed that spermine and the two toxins bind to the closed and open channel conformations of QUIS-R at a site near the intracellular opening of the channel gated by this receptor.

Synthesis and binding of [125I2]philanthotoxin-343, [125I2]philanthotoxin-343-lysine, and [125I2]philanthotoxin-343-arginine to rat brain membranes.

125I2-iodinated philanthotoxin-343 (PhTX-343), [125I2]PhTX-343-arginine, and [125I2]PhTX-343-lysine were synthesized and evaluated as probes for glutamate receptors in rat brain synaptic membranes. It was found that these probes were not specific for the glutamate receptors but may be useful for investigating the polyamine binding site. Filtration assays with Whatman GF/B fiber glass filters were unsuitable because the iodinated PhTX-343 analogues exhibited high nonspecific binding to the filters, thus hindering detection of specific binding to membranes. When binding was measured by a centrifugal assay, [125I2]PhTX-343-lysine bound with low affinity (KD = 11.4 +/- 2 microM) to a large number of sites (37.2 +/- 9.1 nmol/mg of protein). The binding of [125I2]PhTX-343-lysine was sensitive only to the polyamines spermine and spermidine, which displaced [125I2]PhTX-343-lysine with Ki values of (3.77 +/- 1.4) x 10(-5) M and (7.51 +/- 0.77) x 10(-5) M, respectively. The binding was insensitive to glutamate receptor agonists and antagonists. Binding results with [125I2]PhTX-343-arginine were similar to those of [125I2]-PhTX-343-lysine. Considering the high number of toxin binding sites (10000-fold more than glutamate) in these membranes and the insensitivity of the binding to almost all drugs that bind to glutamate receptors, it is evident that most of the binding observed is not to glutamate receptors. On the other hand, PhTX analogues with photoaffinity labels may be useful in the isolation/purification of various glutamate and nicotinic acetylcholine receptors; they could also be useful in structural studies of receptors and their binding sites.

Inactivation of a quisqualate-sensitive glutamate receptor by photosensitive analogues of philanthotoxin.

Three photosensitive, synthetic analogues of delta-philanthotoxin (PhTX-433) have been tested on the locust, excitatory nerve-muscle system. At 10(-9) M they inhibit reversibly the postjunctional currents (EPSCs) recorded from muscle fibres during motor nerve stimulation, mainly by non-competitively antagonizing postjunctional quisqualate-sensitive glutamate receptors (Quis-R), probably through open channel block. This use-dependent antagonism is characteristic of the philanthotoxins. When the preparation was irradiated with 270 nm U.V. during toxin application and nerve stimulation the EPSCs were inhibited irreversibly. Irradiation of the preparation alone or in the presence of philanthotoxins (e.g. PhTX-433) which are not photosensitive did not lead to irreversible inhibition of the EPSC. It is concluded that the three photosensitive toxins bind covalently to Quis-R in its open channel conformation during U.V. irradiation, thereby irreversibly blocking the channel gated by this receptor.

Subacute inhalation toxicity study of an ice-nucleation-active Pseudomonas syringae administered as a respirable aerosol to rats.

The inhalation toxicity of a commercial sample of an ice-nucleation-active Pseudomonas syringae (strain 31a) was evaluated by repetitively exposing rats to about 700 mg/m3 of an aerosol consisting of a suspension of 0.0008, 0.4 or 0.8 g/l of bacteria in water for 2 h per day, 5 days per week for 13-14 exposures. No mortality, moribundity or biologically significant differences in clinical signs, body weight, food consumption or clinical pathology were observed. Animals tested at 500 times (0.4 g/l) and 1000 times (0.8 g/l) the recommended ice-nucleation concentration (0.0008 g/l) exhibited concentration-dependent increased lung weights. Several animals exhibited enlarged tracheobronchial lymph nodes. The pulmonary responses observed are considered compatible with a mild irritant reaction. There was no evidence of bacterial infection. Animals tested at a concentration typical for the discharge mouth of a snow gun (0.0008 g/l) demonstrated no significant biological effect.

Structure-activity relationships of philanthotoxin analogs and polyamines on N-methyl-D-aspartate and nicotinic acetylcholine receptors.

The effects of varying the structure of philanthotoxin (PhTX) were investigated on binding of the channel blockers: [3H]perhydrohistrionicotoxin (H12-HTX) to the nicotinic acetylcholine receptor (nACh-R) of Torpedo electric organ and [3H]MK-801 [( 3H]-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine maleate) to the N-methyl-D-aspartate receptor (NMDA-R) of rat brain cortex. The four moieties of PhTX (tyrosine, butyrate, spermine and the terminal amino group) were modified or conjugated resulting in 36 compounds. Although the potencies of the PhTX analogs on both receptors were higher with increasing lipophilicity and the polyamine chain length, there was considerable divergence between the two receptors' channels in the structural activity requirements for blockade by PhTX analogs. A major difference was the more critical role of the amine terminal for inhibition of the nACh-R than the NMDA-R, whereas the reverse might be true for the tyrosine moiety. The potency range of PhTX analogs on [3H]H12-HTX binding was 1070, but only 21 on [3H]MK-801 binding. Adding a lysine or arginine onto the spermine moiety increased the compound's potency on the nACh-R with little effect on the NMDA-R. Because spermine is a component of PhTX, the effects of five polyamines were also studied. Spermine and spermidine potentiated [3H]MK-801 binding, whereas putrescine, cadeverine and agmatine inhibited it. In presence of glutamate, higher concentrations of all polyamines inhibited [3H]MK-801 binding. On the nACh-R, spermine, spermidine and agmatine inhibited [125I]alpha-bungarotoxin and also [3H]H12-HTX binding in presence of carbamylcholine. The complex nature of PhTX interactions with the two receptors suggests that PhTX may bind to two sites: an external polyamine binding site and a channel binding site.

Fate of Ice Nucleation-Active Pseudomonas syringae Strains in Alpine Soils and Waters and in Synthetic Snow Samples.

The stability of the ice nucleation activity (INA) and viability of INA Pseudomonas syringae 31a, used as an ice nucleator in the manufacture of synthetic snow, was determined in snow. The viability of P. syringae 1-2b, a rifampin-resistant mutant selected from strain 31a to improve recovery from test samples, was determined in laboratory tests of three alpine soil and water samples from three different sources. Snow samples were exposed to environmental conditions or held in darkness at -20 degrees C. Samples of soil and water were maintained in darkness at 0, 7.5, or 15 degrees C. Parent strain 31a INA decreased significantly (>99.0%) in snow exposed to sunlight and freeze-thaw, while the INA of the cell population in snow held in darkness at -20 degrees C remained essentially unchanged. No viable strain 31a was detected in snow exposed to the environment after 7 days, while the viability of strain 31a in snow held in darkness at -20 degrees C decreased to <3% of the original inoculation at the test conclusion. Mutant strain 1-2b viability was undetectable or had decreased significantly 19 days postinoculation in soil samples held at 0 or 15 degrees C. In contrast, 1-2b viability remained detectable at low levels for the duration of the test in soils held at 7.5 degrees C. The 1-2b population demonstrated a significantly longer half-life in peatlike soil than in the loam soils tested. The rate of decrease in 1-2b viability was essentially the same in the three alpine water samples tested with respect to water temperature and sample location.

Spermine and philanthotoxin potentiate excitatory amino acid responses of Xenopus oocytes injected with rat and chick brain RNA.

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin, delta-philanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using voltage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A+)RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10(-11)-10(-7) M) of PhTX-343 and by 10(-5)-10(-4) M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentrations of PhTX-343 and spermine also potentiated the responses to nicotine and gamma-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate receptor.

Structure-activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors.

Fifty-two analogues of the wasp toxin, philanthotoxin-433, have been synthesized and tested on a glutamatergic, nerve-muscle preparation from locust leg. Reduction in amplitude of the neurally-evoked muscle twitch was used to construct dose-inhibition relationships from which IC50S were estimated. The most active analogues were characterized by one or more of the following: increased hydrophobicity of aromatic and tyrosyl regions; an increased number of protonated groups in the polyamine region; a guanidinium instead of a spermine terminal amino moiety. The incorporation of a butyl side-group in the polyamine also enhanced potency. These results are explained on the basis of the known non-competitive antagonistic blockage by philanthotoxin-433 of the channel gated by postjunctional glutamate receptors when the channel is open.

Philanthotoxins. A mass spectrometric investigation.

A method employing liquid secondary-ion mass spectrometry (SIMS) in conjunction with metastable-ion measurements (linked scanning at constant B/E) to obtain sequence-specific information for three synthetic polyamine isomers was developed. The normal liquid SIMS spectra gave molecular weight information, but important sequence ions were of low intensity or obscured by the background. The metastable-ion spectra contained important fragment ions in particular due to cleavage along the polyamine chain. One of the three synthetic isomers was identical with a toxin present in the venom of the digger wasp. In conjunction with nuclear magnetic resonance spectroscopic studies, this should be a powerful method for the structural characterization of other closely related toxins present in the venom of this wasp.

Serologic and protective characterization of Moraxella bovis pili.

This study was conducted to determine the protective nature of purified M. bovis EPP 63 pili in controlling experimentally induced Infectious Bovine Keratoconjunctivitis, and to determine antigenic similarity of pili isolated from various M. bovis isolates. Ten calves were vaccinated twice, 28 days apart, with 5.0 mg (protein) EPP 63 purified pili. Ten calves were maintained as non-vaccinated controls. All calves were exposed to ultraviolet light prior to challenge. The calves were challenged by instilling approximately 2.0 X 10(8) CFU of EPP 63 piliated organisms into the conjunctival sac. Antisera to respective pili types were prepared by immunizing the rabbits with purified pili from M. bovis strains EPP 63, FLA 64, IBH 68, MED 72 and ATCC 10900. Rabbit serum was evaluated for cross reactivity by enzyme-linked immunosorbent assay (ELISA). Purity of pili preparations was demonstrated on SDS-PAGE gels. Molecular weight of pili subunit was determined to be approximately 20,000 for EPP 63, 19,500 for IBH 68 and ATCC 10900, and 17,500 for FLA 64 and MED 72. One of 10 (10%) calves vaccinated with EPP 63 purified pili, and 6 of 10 (60%) nonvaccinated controls developed IBK, respectively. Average eye scores for vaccinates and controls were 0.05 and 0.85, respectively. Significant cross-reaction was found between EPP 63 and MED 72 pili. FLA 64 and ATCC 10900 were similar; however, antiserum to IBH 68 pili showed some degree of cross reaction with other pili.

Role of Escherichia coli type 1 pilus in colonization of porcine ileum and its protective nature as a vaccine antigen in controlling colibacillosis.

This study was designed to evaluate the role of Escherichia coli type 1 pili in adherence of the organism to porcine small intestines and the efficacy of pili as a vaccine antigen in controlling neonatal colibacillosis. Our results demonstrated that an E. coli phase cloned to express type 1 pili readily attached to the small intestines of colostrum-deprived newborn pigs. Immunofluorescent staining of intestine sections revealed the presence of E. coli expressing type 1 pili only on the brush border, suggesting involvement of type 1 pili in the colonization process. Administration of anti-type 1 serum to newborn pigs prior to challenge reduced the level of gut-associated E. coli sixfold compared with controls. Purified type 1 pilus vaccine induced significant protection against colibacillosis in newborn pigs following challenge with E. coli expressing type 1 pili. Pigs born to vaccinated gilts scoured less and gained more weight than pigs born to control gilts. Our results demonstrate that type 1 pili are a virulence factor, as well as an effective vaccine antigen.

Comparison of an infective avirulent and canine virulent Bordetella bronchiseptica.

Two Bordetella bronchiseptica isolates, S-55 and D-2, were evaluated in dogs for inducement of (i) infection, (ii) clinical bordetellosis, and (iii) histopathologic changes on tracheal and bronchiole tissues. Further, each isolate was characterized for variance in (i) toxicity for mice and (ii) intracellular proteins. Both S-55 and D-2 were detectable in test dog groups during the 26-day test period, although 545 times more D-2 was recovered than was S-55. In dogs inoculated with D-2, clinical infectious tracheobronchitis appeared in 4 days and continued for 22 days. Bordetellosis was not observed in dogs given S-55 or in noninoculated dogs. Tracheal and bronchiole tissues from dogs inoculated with the S-55 and D-2 isolates were microscopically examined for lesions. Dogs inoculated with S-55 did not have tracheal or bronchiole lesions. Lesions were not observed in noninoculated dogs. Dogs inoculated with D-2 had marked lesions in the tracheal and bronchiole tissues. The D-2 whole cells were an average 4.8 times as lethal as S-55 whole cells in mice (given intraperitoneal inoculation), whereas cell-free culture supernatants from S-55 and D-2 isolates were nontoxic. Cell-free sonicated extracts of S-55 and D-2 proved toxic to mice (intraperitoneal inoculation), but after the extracts were heated at 56 C for 30 minutes, both were nontoxic. Intracellular proteins of approximately 116,000 and 44,000 daltons were found in higher concentration in D-2 cells than in S-55 cells.

Canine infectious tracheobronchitis: effects of an intranasal live canine parainfluenza-Bordetella bronchiseptica vaccine on viral shedding and clinical tracheobronchitis (kennel cough).

A modified-live intranasal (IN) canine parainfluenza (CPI)-virus Bordetella bronchiseptica vaccine was evaluated in dogs for efficacy against laboratory-induced canine infectious tracheobronchitis. The comparative efficacies of IN and parenteral administrations of the CPI virus fraction were also evaluated. The frequency and duration of clinical tracheobronchitis, blood serum agglutination titer, humoral antibody response, and duration of CPI virus and B bronchiseptica shedding were measured. Group A dogs were vaccinated subcutaneously or IM with an experimental CPI vaccine and challenge exposed with CPI virus. Group B dogs were vaccinated IN with avirulent CPI virus-B bronchiseptica live antigens and challenge exposed with virulent CPI virus and virulent B bronchiseptica. The IN vaccination (group B) significantly reduced (P less than or equal to 0.001) the occurrence of clinical tracheobronchitis by 96%. The combined challenge exposure of virulent CPI and virulent B bronchiseptica produced a synergistic enhancement of the clinical signs of kennel cough. The percentage of days after challenge exposure that virus shedding was detected for controls equaled 70% as compared with 50% and only 1% for parenterally and IN vaccinated dogs, respectively. Isolation of virulent B bronchiseptica microorganisms was reduced 89% in dogs vaccinated IN compared to controls. The geometric mean humoral antibody titers to CPI virus after 2 parenteral vaccinations and 1 IN vaccination were 1:43 and 1:34, respectively.

Intranasal vaccination of dogs with liver avirulent Bordetella bronchiseptica: correlation of serum agglutination titer and the formation of secretory IgA with protection against experimentally induced infectious tracheobronchitis.

Dogs inoculated intranasally with a live avirulent Bordetella bronchiseptica vaccine were monitored for the development of resistance to experimentally induced infectious tracheobronchitis (canine cough). Dogs were challenge exposed with a virulent strains of B bronchiseptica at various times after they were vaccinated. Clinical protection was detectable as early as 48 hours. At postvaccination days 4, 5, and 14, 56%, 83%, and 95% protection was observed. Humoral immunoglobulin (Ig) titers ranged from 1:8.6 on day 0 to 1:147 on postvaccination day 21. In the monitoring of B bronchiseptica-specific secretory IgA by indirect immunofluorescence, titers appeared as early as day 4 after vaccination. The IgA titers ranged from 1:16 on day 4 to 1: 1,024 on day 21. The appearance of IgA titers correlated with the development of resistance to clinical infection.