In silico drug discovery: solving the "target-rich and lead-poor" imbalance using the genome-to-drug-lead paradigm.

Advances in genomics, proteomics, and structural genomics have identified a large number of protein targets. Virtual screening has gained popularity in identifying drug leads by computationally screening large numbers of chemicals against experimentally determined protein targets. In that context, there continues to be a "target-rich and lead-poor" imbalance, reflecting an insufficiency of chemists pursuing drug discovery in academia, the challenge of engaging more chemists in this area of research, and a paucity of available protein target structures. This imbalance in manpower and structural information can be ameliorated, in part, by adapting a "genome-to-drug-lead" approach, in which chemicals can be virtually screened against computer-predicted protein targets, within the context of the US National Science Foundation's petascale computing initiative. This approach offers a solution to reduce manpower requirements for more chemists to experimentally search for drug leads, which represent one of the greatest limitations to drug discovery and better exploits the extensive availability of drug targets at the gene level, ultimately improving the success of moving discoveries from the laboratory to the patient.

Novel nerve-agent antidote design based on crystallographic and mass spectrometric analyses of tabun-conjugated acetylcholinesterase in complex with antidotes.

Organophosphorus compound-based nerve agents inhibit the essential enzyme acetylcholinesterase (AChE) causing acute toxicity and death. Clinical treatment of nerve-agent poisoning is to use oxime-based antidotes to reactivate the inhibited AChE. However, the nerve agent tabun is resistant to oximes. To design improved oximes, crystal structures of a tabun-conjugated AChE in complex with different oximes are needed to guide the structural modifications of known antidotes. However, this type of structure is extremely challenging to obtain because both deamidation of the tabun conjugate and reactivation of AChE occur during crystallographic experiments. Here we report, for the first time, the crystal structures of Ortho-7 and HLö-7 in complex with AChE that is conjugated to an intact tabun. These structures were determined by our new strategy of combining crystallographic and mass spectrometric analyses of AChE crystals. The results explain the relative reactivation potencies of the two oximes and offer insights into improving known medical antidotes.

East meets West in the search for Alzheimer's therapeutics - novel dimeric inhibitors from tacrine and huperzine A.

Alzheimer's disease (AD) is linked to cholinergic deficiency and the overactivation of glutamate receptors. The acetylcholinesterase (AChE) inhibition treatment approach has produced the most encouraging results in clinical practice, and memantine, a moderate antagonist of N-methyl-D-aspartate (NMDA) receptors, has been approved for treating AD. However, AChE inhibitors have limited success as they only improve memory in mild dementia but cannot stop the process of neurodegeneration; while memantine possesses neuroprotective effects only with a little ability in memory enhancement. There has been a major rush among neuroscience research institutions and pharmaceutical firms worldwide to search for safer and more effective therapeutic agents for AD. The novel dimers, derived from tacrine and the fragment of huperzine A (HA'), have been demonstrated to be potent and selective reversible inhibitors of AChE. Bis(7)-tacrine, bis(12)-hupyridone (E12E) and HA'(10)-tacrine, are representatives of three series of novel dimers. According to the preclinical studies, these compounds have been shown to have low toxicity and high efficacy for improving cognitive deficits in several animal models. More interestingly, bis(7)-tacrine, similar to memantine, prevents glutamate-induced neurotoxicity by moderately blocking glutamate receptor NMDA subtype. Furthermore, bis(7)-tacrine, as well as E12E, possesses multiple neuroprotective effects in vitro and in vivo. Taking together, these dimeric AChE inhibitors, especially bis(7)-tacrine, E12E and HA'(10)-tacrine, may provide beneficial effects in AD and other neurodegenerative diseases.

Flavopiridol binds to duplex DNA.

Flavopiridol, the first potent cyclin-dependent kinase inhibitor to enter clinical trials, was recently found to be cytotoxic to noncycling cells. The present studies were performed to examine the hypothesis that flavopiridol, like several other antineoplastic agents that kill noncycling cells, might also interact with DNA. Consistent with this possibility, treatment of A549 human lung cancer cells with clinically achievable concentrations of flavopiridol resulted in rapid elevations of the DNA damage-responsive protein p53. In further studies, the binding of flavopiridol to DNA was examined in vitro by four independent techniques. Absorption spectroscopy revealed that addition of DNA to aqueous flavopiridol solutions resulted in a red shift of the flavopiridol lambda(max) from 311 to 344 nm, demonstrating an isosbestic point typical of changes seen with DNA-binding compounds. Reverse-phase high-performance liquid chromatography demonstrated that flavopiridol binds to genomic DNA to a similar extent as ethidium bromide and Hoechst 33258. Nuclear magnetic resonance spectroscopy revealed that DNA caused extreme broadening of flavopiridol 1H nuclear magnetic resonance signals that could be reversed by addition of ethidium bromide or by DNA melting, suggesting that flavopiridol binds to (and likely intercalates into) duplex DNA. Equilibrium dialysis demonstrated that the equilibrium dissociation constant of the flavopiridol-DNA complex (5.4+/-3.4 x 10(-4) M) was in the same range observed for binding of the intercalators doxorubicin and pyrazoloacridine to DNA. Molecular modeling confirmed the feasibility of flavopiridol intercalation into DNA and analysis of the effects of flavopiridol in the National Cancer Institute tumor cell line panel using the COMPARE algorithm demonstrated that flavopiridol most closely resembles cytotoxic antineoplastic intercalators. Collectively, these data suggest that DNA might be a second target of flavopiridol, providing a potential explanation for the ability of this agent to kill noncycling cancer cells.

Successful molecular dynamics simulation of the zinc-bound farnesyltransferase using the cationic dummy atom approach.

Farnesyltransferase (FT) inhibitors can suppress tumor cell proliferation without substantially interfering with normal cell growth, thus holding promise for cancer treatment. A structure-based approach to the design of improved FT inhibitors relies on knowledge of the conformational flexibility of the zinc-containing active site of FT. Although several X-ray structures of FT have been reported, detailed information regarding the active site conformational flexibility of the enzyme is still not available. Molecular dynamics (MD) simulations of FT can offer the requisite information, but have not been applied due to a lack of effective methods for simulating the four-ligand coordination of zinc in proteins. Here, we report in detail the problems that occurred in the conventional MD simulations of the zinc-bound FT and a solution to these problems by employing a simple method that uses cationic dummy atoms to impose orientational requirement for zinc ligands. A successful 1.0 ns (1.0 fs time step) MD simulation of zinc-bound FT suggests that nine conserved residues (Asn127alpha, Gln162alpha, Asn165alpha, Gln195alpha, His248beta, Lys294beta, Leu295beta, Lys353beta, and Ser357beta) in the active site of mammalian FT are relatively mobile. Some of these residues might be involved in the ligand-induced active site conformational rearrangement upon binding and deserve attention in screening and design of improved FT inhibitors for cancer chemotherapy.

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases.

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery of a new inhibitor lead of adenovirus proteinase: steps toward selective, irreversible inhibitors of cysteine proteinases.

Using the computer docking program EUDOC, in silico screening of a chemical database for inhibitors of human adenovirus cysteine proteinase (hAVCP) identified 2,4,5,7-tetranitro-9-fluorenone that selectively and irreversibly inhibits hAVCP in a two-step reaction: reversible binding (Ki = 3.09 microM) followed by irreversible inhibition (ki = 0.006 s(-1)). The reversible binding is due to molecular complementarity between the inhibitor and the active site of hAVCP, which confers the selectivity of the inhibitor. The irreversible inhibition is due to substitution of a nitro group of the inhibitor by the nearby Cys122 in the active site of hAVCP. These findings suggest a new approach to selective, irreversible inhibitors of cysteine proteinases involved in normal and abnormal physiological processes ranging from embryogenesis to apoptosis and pathogen invasions.

A new form of antiviral combination therapy predicted to prevent resistance from arising, and a model system to test it.

Combination therapy in the treatment of viral infections in which, for example, three different drugs against three different targets on three independent proteins are administered, has been highly successful clinically. However, it is only a matter of time before a virus will arise resistant to all three drugs, because the mutations leading to drug resistance are independent of each other. But, what if the mutations leading to drug resistance are not independent of each other, but confer some cost to the virus? If the cost is too great, than resistance may not arise. To impose such a cost in the clinical treatment of viral infections, we propose a new form of combination therapy. Here, three different drugs against three different targets on the same virus-coded protein are administered. If the physiological functions of the three different target sites are not independent of each other, then, a mutation at one site may alter the physiological functions at the other sites. We present a model system in which to test the efficacy of this new form of triple combination therapy. Human adenovirus has a virus-coded proteinase that is essential for the synthesis of infectious virus. It contains an active site and two cofactor binding sites; the functions of the active site are dependent upon the cofactors interacting with their binding sites. We describe how to obtain drugs against the three different sites.

In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier.

Animal studies with neurotensin (NT) directly injected into brain suggest that it has pharmacological properties similar to those of antipsychotic drugs. Here, we present radioligand binding data for some novel hexapeptide analogs of NT(8-13) at the molecularly cloned rat and human neurotensin receptors (NTR-1), along with behavioral and physiological effects of several of these peptides after intraperitoneal (i.p.) administration in rats. One unique analog, NT66L, which had high affinity (0.85 nM) for the molecularly cloned rat neurotensin receptor (NTR-1), caused a drop in body temperature and antinociception at doses as low as 0.1 mg/kg after i.p. injection. At 30 min post-injection, the ED50 for NT66L-induced hypothermia (rectal temperature) and antinociception (hot plate test) was 0.5 and 0.07 mg/kg, respectively. At a dose of 1 mg/kg i.p., NT66L caused 100% of the maximum possible effect for antinociception for up to 2 h after administration. At this dose body temperature lowering was greater than -2.5 degrees C from 20 to 120 min after i.p. administration. These results in animals suggest that NT66L has agonist properties at NTR-1 in vivo after extracranial administration and provide support for its further study in behavioral tests predictive of neuroleptic activity.

Successful virtual screening of a chemical database for farnesyltransferase inhibitor leads.

Virtual screening of chemical databases is an emerging approach in drug discovery that uses computers to dock chemicals into the active site of a drug target to identify leads through evaluation of binding affinities of the chemicals. However, there are concerns about the validity and scope of the reported virtual screens due to lack of studies to show that randomly selected chemicals are not equally active and due to the fact that metalloproteins were rarely used as drug targets. We have performed a virtual screening of a chemical database to identify prototypic inhibitors of farnesyltransferase (FT) with zinc present in the active site. Among the 21 compounds identified by computers, four inhibited FT in vitro with IC(50) values in the range from 25 to 100 microM. The most potent inhibitor also inhibited FT in human lung cancer cells. In contrast, none of 21 randomly selected compounds have an IC(50) lower than 100 microM. The results demonstrate the validity of virtual screening and the feasibility of applications of this approach to metalloprotein drug targets, such as matrix metalloproteinases, farnesyltransferase, and HIV-1 integrase, for the treatments of cardiovascular diseases, cancers, and AIDS.

Heterodimeric tacrine-based acetylcholinesterase inhibitors: investigating ligand-peripheral site interactions.

Dimeric acetylcholinesterase (AChE) inhibitors containing a single 9-amino-1,2,3,4-tetrahydroacridine (tacrine) unit were constructed in an effort to further delineate structural requirements for optimal binding to the AChE peripheral site. Basic amines of differing hydrophobicity were selected as peripheral site ligands, and in each case, improvements in inhibitory potency and selectivity were seen relative to tacrine itself. AChE IC(50) values of the optimum dimers decrease significantly as the peripheral site ligand was permuted in the series ammonia > dimethylamine > 4-aminopyridine > 4-aminoquinoline > tacrine. Calculated desolvation free energies of the optimum dimers match the trend in IC(50) values, suggesting the importance of ligand hydrophobicity for effective cation-pi interaction with the peripheral site.

Synthesis and biological activity of 8a-phenyldecahydroquinolines as probes of PCP's binding conformation. A new PCP-like compound with increased in vivo potency.

The synthesis and chemical resolution of cis- and trans-fused 8a-phenyldecahydroquinolines 3 and 4 are described together with the affinity of the four optically pure compounds for the PCP recognition site of the NMDA receptor complex. These compounds were also evaluated for their antagonistic effects on cGMP levels in male Swiss Webster mice, and (-)-4 was found to exhibit in vivo potency comparable to that of MK-801. The results of the binding studies are interpreted in terms of a preferred orientation of PCP's N-H bond in binding to its NMDA receptor-associated recognition site.

Synthesis and evaluation of novel aldose reductase inhibitors: Effects on lens protein kinase Cgamma.

PURPOSE: To synthesize novel aldose reductase inhibitors (ARI) that will normalize losses in protein kinase Cgamma (PKCgamma) observed during diabetes and galactosemia. METHODS: ARI were synthesized as tricyclic pyrones 1-6 (HAR-1 through HAR-6) from 3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran and (5aS,7S)-7-isopropenyl-3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran and were tested by inhibition of aldose reductase enzyme activity in vitro and by inhibition of polyol formation in lens epithelial cells in culture. Identified compounds were further tested in galactosemic rat lens in vivo for (a) normalized PKCgamma levels by Western blot, (b) reduction of phosphorylation of the gap junction protein Cx46 by analyses of co-immunoprecipitated proteins, and (c) by normalization of gap junction activity as measured by dye transfer. RESULTS: HAR-1 (1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran-3-acetic acid) was identified as an ARI with IC50 for aldose reductase inhibition at 2 nM. Polyol accumulation in lens epithelial cells was reduced by 80% at 10 microM. Rats fed 40% galactose for 9 days had an 80% reduction in PKCgamma levels which were normalized by HAR-1 at 100 mg/kg/day, fed orally. Phosphorylation of Cx46 was increased by 50% and this was normalized in HAR-1 treated rats (6 day treatment). Gap junction activity of galactosemic rats was reduced by 55% and this was normalized by HAR-1 in six day-treated rats. CONCLUSIONS: HAR-1 is a novel ARI which normalized losses of PKCgamma, changes in Cx46 phosphorylation, and gap junction activity.

Effects of bis(7)-tacrine, a novel anti-Alzheimer's agent, on rat brain AChE.

The anticholinesterase effects of bis(7)-tacrine were compared with tacrine in vitro and in vivo. Based on IC50 ratios, the dimeric analog bis(7)-tacrine was, in a reversible manner, up to 150-fold more potent and 250-fold more selective than tacrine for acetylcholinesterase (AChE) over butyrylcholinesterase (BChE). Following a single oral administration, both bis(7)-tacrine and tacrine produced dose-dependent inhibitions of AChE in rat brain, but bis(7)-tacrine exhibited higher efficacy and AChE/BChE selectivity than tacrine. The anti-AChE efficacy of bis(7)-tacrine was quite similar following an oral or i.p. administration, but tacrine showed much lower efficacy when administered orally than when given i.p. These findings suggest bis(7)-tacrine, a highly potent and selective inhibitor of AChE, can probably be used as an improved drug in the palliative treatment of AD.

Bis(7)-tacrine, a novel dimeric AChE inhibitor, is a potent GABA(A) receptor antagonist.

Heptylene-linked bis-(9-amino-1,2,3,4-tetrahydroacridine) (bis(7)-tacrine) is a potential palliative therapeutic agent for Alzheimer's disease (AD), on the basis of its superior acetylcholinesterase (AChE) inhibition and memory-enhancing potency relative to tacrine. In this study we report that bis(7)-tacrine exhibits a potentially complementary central nervous system action, antagonism of GABA(A) receptor function. Bis(7)-tacrine displaced [3H]muscimol from rat brain membranes with an apparent Ki of 6.0 microM; tacrine and physostigmine were shown to be 18 and 170 times less potent, respectively. In whole-cell patch-clamp recordings, bis(7)-tacrine inhibited GABA-induced inward current with an IC50 of 5.6 microM, and shifted the GABA concentration-response curve to the right in a parallel manner. These results suggest that bis(7)-tacrine is a competitive antagonist of the GABA(A) receptor.

Protection against ischemic injury in primary cultured mouse astrocytes by bis(7)-tacrine, a novel acetylcholinesterase inhibitor [corrected].

The effects of bis(7)-tacrine, a novel acetylcholinesterase inhibitor, on ischemia-induced cell death and apoptosis were investigated in primary cerebral cortical astrocytes of mice. Following a 6 h in vitro ischemic incubation of the cultures, a marked decrease in the percentage of viable cells was observed by lactate dehydrogenase (LDH) release assay. Furthermore, using bisbenzimide staining, we determined that approximately 65% of the cells underwent apoptosis. Treatment with bis(7)-tacrine (1-10 nM) during ischemic incubation effectively inhibited the ischemia-induced apoptosis, as demonstrated by morphological and biochemical tests. Our results demonstrated that bis(7)-tacrine could protect astrocytes against ischemia-induced cell injury, indicating that the drug might be beneficial for the treatment of vascular dementia, in addition to Alzheimer's disease.

[Long-term surveillance after basic elimination of bancroftian filariasis].

The longitudinal and cross-sectional systemic surveillance have been conducted for 9-11 consecutive years in six counties (cities) of Guangxi Zhuang Autonomous Region after basic elimination of bancroftian filariasis. The two different control regimens had been used with DEC selective treatment followed by mass treatment of all persons and selective treatment followed by taking DEC medicated salt. During the former 6 years, residual microfilaremia cases could still be detected; whereas during the latter 5 years, no microfilaremia cases could be detected at all. The natural infection of vector mosquitoes showed negative. The positive rate of antibody in the populations was reduced to 1.4-5.5% detected by IFAT, reaching to the level of local non-endemic areas. The result indicated that the transmission of filariasis in these areas has been blocked. The authors suggested that a period of 10 years might be appropriate for surveillance after basic elimination of bancroftian filariasis.

Molar pregnancy with false negative urine hCG: the hook effect.

Urine pregnancy tests are widely used in emergency departments as the first screening test for patients of reproductive age presenting with gynaecological problems in order to rule out pregnancy. Conditions such as complete molar pregnancy, which produces large amounts of beta human chorionic gonadotropin (beta-hCG), may cause a false negative result due to an oversaturation of the assay system, known as the "hook effect". We report a case where the exclusion of pregnancy by urine testing led to the initial misdiagnosis of a molar pregnancy as a degenerative fibroid. Physicians need to be reminded of the possibility of false negative results with this commonly used test. Negative or inconclusive results in patients with a high suspicion of pregnancy should be further evaluated by serum quantification of beta-hCG and appropriate sample dilution.

Successful molecular dynamics simulation of two zinc complexes bridged by a hydroxide in phosphotriesterase using the cationic dummy atom method.

I report herein two 2.0 ns (1.0 fs time step) MD simulations of two zinc complexes bridged by a hydroxide in phosphotriesterase (PTE) employing the nonbonded method and the cationic dummy atom method that uses virtual atoms to impose orientational requirement for zinc ligands. The cationic dummy atom method was able to simulate the four-ligand coordination of the two zinc complexes in PTE. The distance (3.39 +/- 0.07A) between two nearby zinc ions in the time-average structure of PTE derived from the MD simulation using the cationic dummy atoms matched that in the X-ray structure (3.31 +/- 0.001A). Unequivocally, the time-average structure of PTE was able to fit into the experimentally determined difference electron density map of the corresponding X-ray structure. The results demonstrate the practicality of the cationic dummy atom method for MD simulations of zinc proteins bound with multiple zinc ions. In contrast, a 2.0 ns (1.0 fs time step) MD simulation using the nonbonded method revealed a striking difference in the active site between the X-ray structure and the time-average structure that was unable to fit into the density map of PTE. The results suggest that caution should be used in the MD simulations using the nonbonded method.