Prediction of small-molecule binding to cytochrome P450 3A4: flexible docking combined with multidimensional QSAR

The inhibition of cytochrome P450 3A4 (CYP3A4) by small molecules is a major mechanism associated with undesired drug-drug interactions, which are responsible for a substantial number of late-stage failures in the pharmaceutical drug-development process. For a quantitative prediction of associated pharmacokinetic parameters, a computational model was developed that allows prediction of the inhibitory potential of 48 structurally diverse molecules. Based on the experimental structure of CYP3A4, possible binding modes were first sampled by using automated docking (Yeti software) taking protein flexibility into account. The results are consistent with both X-ray crystallographic data and data from metabolic studies. Next, an ensemble of energetically favorable orientations was composed into a 4D dataset for use as input for a multidimensional QSAR technique (Raptor software). A dual-shell binding-site model that allows an explicit induced fit was then generated by using hydrophobicity scoring and hydrogen-bond propensity. The simulation reached a cross-validated r2 value of 0.825 and a predictive r2 value of 0.659. On average, the predicted binding affinity of the training ligands deviates by a factor of 2.7 from the experiment; those of the test set deviate by a factor of 3.8 in Ki.     

Inhibition of Golgi mannosidase II with mannostatin A analogues: synthesis, biological evaluation, and structure-activity relationship studies

Mannostatin and aminocyclopentitetrol analogues with various substitutions at the amino function were synthesized. These compounds were tested as inhibitors of human Golgi and lysosomal alpha-mannosidases. Modification of the amine of mannostatin had only marginal effects, whereas similar modifications of aminocyclopentitetrol led to significantly improved inhibitors. Ab initio calculations and molecular docking studies were employed to rationalize the results. It was found that mannostatin and aminocyclopentitretrol could bind to Golgi alpha-mannosidase II in a similar mode to that of the known inhibitor swainsonine. However, due to the flexibility of the five-membered rings of these compounds, additional low-energy binding modes could be adopted. These binding modes may be relevant for the improved activities of the benzyl-substituted compounds. The thiomethyl moiety of mannostatin was predicted to make favorable hydrophobic interactions with Arg228 and Tyr727 that would possibly account for its greater inhibitory activity.     

Discovery and structure-activity relationship studies of a unique class of HIV-1 integrase inhibitors

HIV-1 integrase (IN) is an essential enzyme for viral replication and a validated target for the development of drugs against AIDS. Currently there are no approved drugs that target IN. However, new IN inhibitors are under clinical investigation. As more IN inhibitors enter human drug trials, there is a growing need for the design of novel lead compounds with diverse structural scaffolds and promising pharmacokinetic properties to counteract the difficulties observed with first-generation IN inhibitors. We have identified a novel class of IN inhibitors through the systematic exploration of structure-activity relationships in a series of linomide analogues. The predicted bound conformation of the most active analogues inside the IN active site also supports the observed structure-activity correlation in this new compound class.     

Design, synthesis, and biological evaluation of 1-[(biarylmethyl)methylamino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ols as potent antifungal agents: new insights into structure-activity relationships

We recently reported the design and synthesis of azole antifungal agents with a focus on modifications to the side chain appended to the propanol group. Herein we have identified a series of new 1-[(biarylmethyl)methylamino] derivatives with broad-spectrum antifungal activities against the most prevalent human pathogenic fungi (Candida spp. and Aspergillus fumigatus). Compounds containing a flexible benzylamine moiety were clearly shown to yield the best antifungal activities, without the need for a hydrogen-bond acceptor substituent directly attached to the para position. We were also able to determine that selected compounds are able to overcome gene overexpression and point mutations that lead to reduced susceptibility or resistance against current treatments, such as fluconazole. As the minor differences observed with small structural modifications cannot be explain with only a three-dimensional model of CYP51, adequate physicochemical parameters must be evaluated in terms of antifungal potency, bioavailability, and toxicity. Therefore, structure-activity relationship studies such as these reveal new insights for the development of future antifungal therapies.     

Nanostructure formation enhances the activity of LPS-neutralizing peptides

Peptides that interact with lipopolysaccharide (LPS) can provide the basis for the development of new antisepsis agents. In this work, several LPS-neutralizing acyl peptides derived from LALF, BPI, and SAP were prepared, structurally characterized, and biologically evaluated. In all cases, peptides with long acyl chains showed greater LPS-neutralizing activities than the original acetylated peptides. Structural analysis of these peptides revealed that N-acylation with long acyl chains promotes the formation of micellar or fibril-like nanostructures, thus proving a correlation between anti-LPS activity and nanostructure formation. The results of this study provide useful structural insight for the future design of new acyl peptides that strongly bind LPS and therefore act as antisepsis drugs. Furthermore, this nanostructure-biological activity correlation can be translated into other therapeutic areas.     

Design, synthesis, and biological activity of urea derivatives as anaplastic lymphoma kinase inhibitors

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.     

GPCR antitarget modeling: pharmacophore models for biogenic amine binding GPCRs to avoid GPCR-mediated side effects

G protein-coupled receptors (GPCRs) form a large protein family that plays an important role in many physiological and pathophysiological processes. However, the central role that the biogenic amine binding GPCRs and their ligands play in cell signaling poses a risk in new drug candidates that reveal side affinities towards these receptor sites. These candidates have the potential to interfere with the physiological signaling processes and to cause undesired effects in preclinical or clinical studies. Here, we present 3D cross-chemotype pharmacophore models for three biogenic amine antitargets: the alpha(1A) adrenergic, the 5-HT(2A) serotonin, and the D2 dopamine receptors. These pharmacophores describe the key chemical features present within these biogenic amine antagonists and rationalize the biogenic amine side affinities found for numerous new drug candidates. First applications of the alpha(1A) adrenergic receptor model reveal that these in silico tools can be used to guide the chemical optimization towards development candidates with fewer alpha(1A)-mediated side effects (for example, orthostatic hypotension) and, thus, with an improved clinical safety profile.     

Conformational analysis of bivalent estrogen receptor ligands: from intramolecular to intermolecular binding

The estrogen receptor binding affinities of bivalent raloxifene ligands tethered by flexible spacers of different lengths have been evaluated in vitro. Two bivalent binding modes, intra- and intermolecular, were hypothesized to explain their different binding properties. The binding affinities of these bivalent ligands in an aqueous environment are influenced by their conformations, which can be determined by 2D NMR and UV spectral methods. Moreover, computer modeling and simulations were performed to explain the binding modes of these bivalent ligands and to estimate the conformational entropy difference between their unbound and bound states. It was found that bivalent ligands tethered by long spacers had weaker binding affinities because of the shielding of the binding moieties that results from their folded conformations; those tethered by short spacers had stronger affinities because they exposed their ligands to the receptor.     

Design, synthesis, and pharmacological evaluation of mefloquine-based ligands as novel antituberculosis agents

Tuberculosis (TB) is presently regarded as one of the most dangerous infective diseases worldwide and one of the major AIDS-associated infections. To shorten the current treatment regimen, there is an urgent need to identify new anti-TB agents which are active against both replicating TB (R-TB) and nonreplicating TB (NRP-TB). Mefloquine, a well-known antimalarial drug was found to possess reasonable activity against NRP-TB, and accordingly, 30 new analogues were synthesized and evaluated for their anti-TB activity against Mycobacterium tuberculosis H(37)Rv. As the target of mefloquine in Mycobacterium tuberculosis remains unknown, we resorted to modifying mefloquine in a variety of chemically convenient ways, which led us in turn to the active hydrazone 10 a. Further modifications of 10 a led to compound 7 f, with an improved anti-TB activity/selectivity profile with both less cytotoxicity and less predicted CNS side effects compared with mefloquine. The clear structure-activity relationships (SARs) derived from this study should facilitate our ultimate goal of identifying improved anti-TB agents.     

Design and stereoselective synthesis of retinoids with ferrocene or N-butylcarbazole pharmacophores that induce post-differentiation apoptosis in acute promyelocytic leukemia cells

New ferrocene and N‐alkylcarbazole retinoids were designed and synthesized stereoselectively in good yields. A number of these synthesized ligands, in particular 2 , 3 , and 11 , were found to exhibit a high RARα activation potential and to effectively induce post‐differentiation apoptosis in NB4 acute promyelocytic leukemia (APL) cells. Increasing the length of the side chain attached to the heterocycle of the carbazole arotinoids creates new opportunities for altered compound catabolism and for fine‐tuning of the apoptosis‐inducing potential of the ligand. In the carbazole series of new retinoids, maximal activity was established for N‐butylcarbazole analogue 11 in all assays (i.e., RARα activation, differentiation induction, and apoptosis induction). Study of the mechanism of apoptosis revealed an activation of initiator caspases‐8 and ‐9, followed by efficient cleavage of effector caspase‐3 on day 6 of treatment. Subsequent induction of a caspase cascade in NB4 cells triggered ultimate leukemic cell death. The selected ligands 2 , 3 , and 11 may provide alternate options for the treatment of APL in cases of life‐threatening ATRA syndrome, resistance, and high toxicity to conventionally used retinoids.     

Design, synthesis, and in vitro antifungal activity of 1-[(4-substituted-benzyl)methylamino]-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ols

As part of our studies focused on the design of 1‐[((hetero)aryl‐ and piperidinylmethyl)amino]‐2‐phenyl‐3‐(1H‐1,2,4‐triazol‐1‐yl)propan‐2‐ols as antifungal agents, we report the development of new extended benzylamine derivatives substituted at the para position by sulfonamide or retrosulfonamide groups linked to alkyl or aryl chains. These molecules have broad‐spectrum antifungal activities not only against Candida spp., including fluconazole‐resistant strains, but also against a filamentous species (A. fumigatus). Concerning fluconazole resistance, selected compounds exhibit the capacity to overcome CDR and ERG11 gene upregulation and to maintain antifungal activity despite a recognized critical CYP51 substitution in C. albicans isolates. Synthesis, investigation of the mechanism of action by sterol analysis in a C. albicans strain, and structure–activity relationships (SARs) are reported.     

Structure-activity studies on the corticotropin releasing factor antagonist astressin, leading to a minimal sequence necessary for antagonistic activity

Corticotropin Releasing Factor (CRF) antagonists are considered promising for treatment of stress-related illnesses such as major depression and anxiety-related disorders. We report here the design, synthesis and biological evaluation of 91 truncated astressin analogues in order to deduce the pharmacophoric amino acid residues. Such truncated peptides may serve as valuable lead structures for the development of new small, non-peptide-based CRF antagonists. N-Terminal truncation of astressin led to active CRF antagonists that are substantially reduced in size and are selectively active at the human CRF receptor type 1 in vitro and in vivo. Subsequently, an alanine scan in combination with further truncated derivatives led to the proposal of a new pharmacophoric model of peptide-based CRF antagonists. It was found that the astressin(27-41)C sequence is the shortest active CRF antagonist. The first eight N-terminal amino acid residues were found to be an important structural determinant and were replaceable by alanine residues, thus enhancing the alpha-helical propensity. A covalent structural constraint is of utmost importance for the preorganization of the C-terminal amino acid residues. The C-terminal heptapeptide sequence, however, was found to be crucial for the antagonistic activity, since substitution or deletion of any residue led to inactive compounds.     

Analysis of metal ion dependence in glmS ribozyme self-cleavage and coenzyme binding

The bacterial glmS ribozyme is mechanistically unique among both riboswitches and RNA catalysts. Its self-cleavage activity is the basis of riboswitch regulation of glucosamine-6-phosphate (GlcN6P) production, and catalysis requires GlcN6P as a coenzyme. Previous work has shown that the coenzyme amine of GlcN6P is essential for glmS ribozyme self-cleavage, as is its protonation state. Metal ions are also essential within the glmS ribozyme core for both structure and function of the ribozyme. Although metal ions do not directly promote catalysis, we show that metal ion identity and the varying physicochemical properties of metal ions have an impact on the rate of glmS ribozyme self-cleavage. Specifically, these studies demonstrate that metal ion identity influences the overall apparent pK(a) of ribozyme self-cleavage, and metal ion binding largely reflects phosphate oxygen affinity. Results suggest that metal ions take alternative roles in supporting the mechanism of catalysis.     

The activation of fibroblast growth factors (FGFs) by glycosaminoglycans: influence of the sulfation pattern on the biological activity of FGF-1

Six synthetic heparin-like oligosaccharides have been used to investigate the effect of the oligosaccharide sulfation pattern on the stimulation of acidic fibroblast growth factor (FGF-1) induced mitogenesis signaling and the biological significance of FGF-1 trans dimerization in the FGF-1 activation process. It has been found that some molecules with a sulfation pattern that does not contain the internal trisaccharide motif, which has been proposed for high affinity for FGF-1, stimulate FGF-1 more efficiently than those with the structure of the regular region of heparin. In contrast to regular region oligosaccharides, in which the sulfate groups are distributed on both sides of their helical three-dimensional structures, the molecules containing this particular sulfation pattern display the sulfate groups only on one side of the helix. These results and the fact that these oligosaccharides do not promote FGF-1 dimerization according to sedimentation-equilibrium analysis, confirm the importance of negative-charge distribution in the activation process and strongly suggest that FGF dimerization is not a general and absolute requirement for biological activity.     

DNA compaction by divalent cations: structural specificity revealed by the potentiality of designed quaternary diammonium salts

DNA interaction with quaternary diammonium dications, R(CH(3))(2)N(+)(CH(2))(n)N(+)(CH(3))(2)R, having various intercharge distances, lengths, and branching, and the chemical nature of the hydrophobic substituents were investigated by fluorescent microscopy and circular dichroism (CD) spectroscopy to reveal their structural specificity for binding to DNA. The conformational behavior of DNA was found to be highly sensitive to the structure of the dications with separated charges. The distance between two ammonium groups greatly influences the compaction activity of the dications. To explain this situation, we proposed a model that demonstrates that the charge density of the dication and the geometric fit between DNA phosphates and the ammonium groups in the dications play an important role in providing efficient DNA collapse. Elongation of the alkyl substituents (R) in the diammonium salts from ethyl to hexyl did not generate any significant alterations in the compaction activities, whereas the branching of substituents caused a drastic decrease in their compaction ability. Based on the results of CD spectroscopy, it was found that the ability of the dications to provoke a DNA transition from the B-form to A-form was also specific: it depended on their intercharge distances and was independent of the length of alkyl substituents.     

Selective human adenosine A3 antagonists based on pyrido[2,1-f]purine-2,4-diones: novel features of hA3 antagonist binding

Based on our previous results on the potent antagonist effect of 1H,3H-pyrido[2,1-f]purine-2,4-diones at the human A(3) adenosine receptor, new series of this family of compounds have been synthesized and evaluated in radioligand binding studies against the human A(1), A(2A), A(2B), and A(3) receptors. A remarkable improvement in potency, and most noticeable, in selectivity has been achieved, as exemplified by the 3-cyclopropylmethyl-8-methoxy-1-(4-methylbenzyl)-1H,3H-pyrido[2,1-f]purine-2,4-dione (10) that combines a very high affinity at hA(3) (K(i)=2.24 nM), with lack of affinity for the A(1), A(2A), and A(2B) receptors. On the basis of the published hA(3) receptor model (PDB 1OEA), molecular modeling studies, including molecular dynamics (MD) simulations, have been performed to depict the binding mode of the 1 H,3H-pyrido[2,1-f]purine-2,4-diones and to justify the selectivity against the other adenosine receptors. These studies have led to novel features of the cavity where our antagonists are bound so that the cavity is lined by the hydrogen-bonded Gln 167-Asn 250 pair and by the highly conserved Phe 168.