Insight into binding mechanisms of inhibitors MKP56, MKP73, MKP86, and MKP97 to HIV-1 protease by using molecular dynamics simulation

HIV-1 protease (PR) has been a significant target for design of potent inhibitors curing acquired immunodeficiency syndrome. Molecular dynamics simulations coupled with molecular mechanics Poisson–Boltzmann surface area method were performed to study interaction modes of four inhibitors MKP56, MKP73, MKP86, and MKP97 with PR. The results suggest that the main force controlling interactions of inhibitors with PR should be contributed by van der Waals interactions between inhibitors and PR. The cross-correlation analyses based on MD trajectories show that inhibitor binding produces significant effect on the flap dynamics of PR. Hydrogen bond analyses indicate that inhibitors can form stable hydrogen bonding interactions with the residues from the catalytic strands of PR. The contributions of separate residues to inhibitor bindings are evaluated by using residue-based free energy decomposition method and the results demonstrate that the CH–π and CH–CH interactions between the hydrophobic groups of inhibitors with residues drive the associations of inhibitors with PR. We expect that this study can provide a significant theoretical aid for design of potent inhibitors targeting PR.     

Discovery of potent and selective spiroindolinone MDM2 inhibitor,RO8994, for cancer therapy

The field of small-molecule inhibitors of protein–protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.     

Prodrug-based design, synthesis, and biological evaluation of N-benzenesulfonylpiperidine derivatives as novel, orally active factor Xa inhibitors

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element. This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC50 = 13 nM) and excellent selectivity over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demonstrated an anticoagulant effect after oral dosing.     

Significant effects of Z-Gln-Val-Val-OME, common sequences of thiol proteinase inhibitors on thiol proteinases

The first studies on a series of the small synthetic thiol proteinase inhibitors, conservative common sequences in several thiol proteinase inhibitors, are described. Among the many interesting findings with synthetic thiol proteinase inhibitors was the observation that the most effective analogue, Z-Gln-Val-Val-Ala-Gly-OMe, whose amino and carboxyl groups were protected with Z and OMe, respectively, showed inhibitory activity on papain and cathepsin B and protected papain from egg cystatin, human low-molecular-weight kininogen and T-kininogen-induced inhibition but not from leupeptin-induced inhibition. Moreover, it was revealed that Z-Gln-Val-Val-OMe was the smallest peptide to exhibit a protective effect on papain.     

Design, synthesis, and bioactivity of novel inhibitors of E. coli aspartate transcarbamoylase

A series of inhibitors of the aspartate transcarbamoylase, an enzyme involved in pyrimidine nucleotide biosynthesis, has been synthesized. These inhibitors are analogues of a highly potent inhibitor of this enzyme, N-phosphonacetyl-L-aspartate (PALA). Analogues have been synthesized with modifications at the alpha- and beta-carboxylates as well as at the aspartate moiety. The ability of these compounds to inhibit the enzyme was evaluated. These studies, with functional group modified PALA derivatives, showed that amide groups can be a useful substitute of the carboxylate in order to reduce the charge on the molecule, and indicate that the relative position of the functional group in the beta-position is more critical than the nature of the functional group. Some of the molecules synthesized here are potent inhibitors of the enzyme.     

Action of major insecticide groups on insect cell lines of the beet armyworm, Spodoptera exigua, compared with larvicidal toxicity

In a first series of experiments, the biological response of a continuous cell line of the beet armyworm, Spodoptera exigua, was tested with different groups of insecticides with different modes of actions: acetylcholinesterase inhibitors, acetycholine receptor agonists, inhibitors and uncouplers of oxidative phosphorylation, site I electron transport inhibitors, gamma-aminobutyric acid receptor inhibitors, chitin synthesis inhibitors, and juvenile hormone analogues. From the concentration response curves, 50% inhibition concentration (IC(50)) values were calculated. The most active compound in vitro was pyridaben with an IC(50) value of 0.0083 ppm. In a second series of experiments, the toxicity of these insecticide groups was determined on third-instar larvae of S. exigua, and lethal concentration with 50% kill (LC(50)) values were used in the evaluation of their in vivo biological activity. Toxicity bioassays showed that lufenuron was the most toxic (LC(50) = 0.098 ppm). To explain the discrepancies in biological responses in vitro with insect cells compared with in vivo conditions with whole third-instar larvae, the significance of different detoxifying enzyme systems was tested. P(450) monooxygenases, esterases, and glutathione S-transferases were measured in third-instar larvae and cells of S. exigua. Data are discussed in terms of the usefulness of insect cell cultures as tools in the screening for novel insecticide actions.     

Novel proteinase inhibitors in seeds of sunflower (Helianthus annuus L.): polymorphism, inheritance and properties

A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC allowed the M _r of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors varied in M _r from about 1,500 to 6,000. Received: 7 March 1999 / Accepted: 18 March 1999     

Natural,semisynthetic and synthetic tyrosinase inhibitors

Tyrosinase plays a pivotal role in the synthesis of melanin pigment synthesis on skin utilizing tyrosine as a substrate. Melanin is responsible for the protection against harmful ultraviolet irradiation, which can cause significant pathological conditions, such as skin cancers. However, it can also create esthetic problems when accumulated as hyperpigmented spots. Various skin-whitening ingredients which inhibit tyrosinase activity have been identified. Some of them, especially ones with natural product origins, possess phenolic moiety and have been employed in cosmetic products. Semi-synthetic and synthetic inhibitors have also been developed under inspiration of the natural inhibitors yet some of which have no phenolic groups. In this review, tyrosinase inhibitors with natural, semi-synthetic and synthetic origins are listed up with their structures, activities and characteristics. Further, a recent report on the adverse effect of a natural melanin synthesis inhibitor which was included in skin-whitening cosmetics is also briefly discussed.     

Novel histone deacetylase inhibitors: design, synthesis, enzyme inhibition, and binding mode study of SAHA-based non-hydroxamates

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) were designed and synthesized as (i) substrate (acetyl lysine) analogues (compounds 3–7), (ii) analogues bearing various functional groups expected to chelate zinc ion (compounds 8–15), and (iii) analogues bearing nucleophilic functional groups which could bind covalently to HDACs (compounds 16–18). In this series, semicarbazide 8b and bromoacetamides 18b,c were found to be potent HDAC inhibitors for non-hydroxamates.     

Benzimidazole design,synthesis, and docking to build selective carbonic anhydrase VA inhibitors

The similarity of human carbonic anhydrase (CA) active sites makes it difficult to design selective inhibitors for one or several CA isoforms that are drug targets. Here we synthesize a series of compounds that are based on 5-[2-(benzimidazol-1-yl)acetyl]-2-chloro-benzenesulfonamide (1a) which demonstrated picomolar binding affinity and significant selectivity for CA isoform five A (VA), and explain the structural influence of inhibitor functional groups to the binding affinity and selectivity. A series of chloro-substituted benzenesulfonamides bearing a heterocyclic tail, together with molecular docking, was used to build inhibitors that explore substituent influence on the binding affinity to the CA VA isoform.     

Design, synthesis, and biological testing of potential heme-coordinating nitric oxide synthase inhibitors

Based on computer modeling of the active site of nitric oxide synthases (NOS), a series of 10 amidine compounds (9-18) was designed including potential inhibitors that involve the coordination of side-chain functional groups with the iron of the heme cofactor. The most potent and selective compound was the methylthio amidine analogue 9, which was more potent than L-nitroarginine with 185-fold selectivity for inhibition of neuronal NOS over endothelial NOS. It also exhibited time-dependent inhibition, but did not involve the mechanism previously proposed for other amidine inhibitors of NOS. None of the compounds, however, exhibited heme-binding characteristics according to absorption spectroscopy.     

Synthesis,SAR, and atropisomerism of imidazolopyrimidine DPP4 inhibitors

The synthesis and SAR of aminomethyl-substituted imidazolopyrimidine DPP4 inhibitors bearing varied pendant aryl groups is described. Compound 1, which exists as a separable mixture of non-interconvertible atropisomers was used as the starting point for investigation. The effects of substituent pattern and type as well as stereochemical effects on inhibitor potency are discussed.     

Structure-activity studies of cyclic ketone inhibitors of the serine protease plasmin: design, synthesis, and biological activity

Three series of cyclic ketone inhibitors were synthesized and evaluated against the serine protease plasmin. Peptide inhibitors that incorporated 3-oxotetrahydrofuran and 3-oxotetrahydrothiophene 1,1-dioxide groups had the highest activities. Alkylamino substituents, which were designed to bind in the S1 subsite of plasmin, were attached to the inhibitors. Compounds 5c and 5g, which incorporated 6-aminohexyl substituents, were found to be optimal and demonstrated IC₅₀ values in the low micromolar range. Incorporating conformationally constrained peptide segments into the inhibitors did not improve their activities.     

Substituent effects on P2-cyclopentyltetrahydrofuranyl urethanes: design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors

The design, synthesis, and biological evaluation of novel C3-substituted cyclopentyltetrahydrofuranyl (Cp-THF)-derived HIV-1 protease inhibitors are described. Various C3-functional groups on the Cp-THF ligand were investigated in order to maximize the ligand-binding site interactions in the flap region of the protease. Inhibitors 3c and 3d have displayed the most potent enzyme inhibitory and antiviral activity. Both inhibitors have maintained impressive activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 3c-bound HIV-1 protease revealed a number of important molecular insights into the ligand-binding site interactions.     

Design, synthesis, and SAR of anthranilamide-based factor Xa inhibitors with improved functional activity

Compound 2 containing an aminomethylbenzoyl moiety as the S4 binding motif was synthesized in order to modulate hydrophlicity of anthranilamide-based factor Xa inhibitors with substituted biphenyl P4 groups. Structure-activity relationship studies around 2 have led to a series of potent factor Xa inhibitors which are highly active in the human plasma-based thrombin generation assay with 2XTG values less than 1 microM. Compound 55 shows strong antithrombotic activity in our rabbit deep vein thrombosis model, and also exhibits good oral bioavailability and a long half life in rats.     

Influence of synthetic peptide inhibitors on the thermal stability of thermitase, a serine proteinase from Thermoactinomyces vulgaris

The influence of chloromethyl ketones and methyl ketones of N-acylated peptides on the thermal denaturation of thermitase was investigated in the presence and the absence of calcium ions. The chloromethyl ketone derivatives are known to react irreversibly with the enzyme, whereas the corresponding methyl ketones are reversible inhibitors. Both groups of inhibitors offer a broad variety of affinity constants. The irreversible inhibition of thermitase causes a marked stabilization against thermal denaturation. On the other hand, the enzyme stability is not influenced by the binding of reversible inhibitors. The stabilizing effect of calcium ions is not dependent on the inhibitor binding. The importance of bivalent interaction (bridge formation) in the active site region of the enzyme for its thermal stability is discussed.     

Effects of Methylated, Organic, and Inorganic Substrates on Microbial Consumption of Dimethyl Sulfide in Estuarine Waters

We examined the effects of a variety of amendments on the consumption of [U-¹⁴C]dimethyl sulfide in a Georgia salt marsh. Methylated compounds, particularly those with dimethyl groups, significantly inhibited dimethyl sulfide consumption, while nonmethylated substrates had little effect. Dimethyl disulfide and dimethyl ether were the most effective inhibitors tested.     

Design,synthesis, and SAR of substituted acrylamides as factor Xa inhibitors

Substituted acrylamides were used as templates that bridge P1 and P4 binding elements, resulting in a series of potent (sub-nanomolar) and selective factor Xa inhibitors. In this template, cis-geometry of P1 and P4 ligands is highly preferred. SAR on the substituting groups, as well as on modification of P1 and P4 moieties is described. Compounds in this series show good in vivo efficacy in animal models.     

ROCK inhibitors 2. Improving potency,selectivity and solubility through the application of rationally designed solubilizing groups

Solubilizing groups have been frequently appended to kinase inhibitor drug molecules when solubility is insufficient for pharmaceutical development. Such groups are usually located at substitution sites that have minimal impact on target activity. In this report we describe the incorporation of solubilizing groups in a class of Rho kinase (ROCK) inhibitors that not only confer improved solubility, but also enhance target potency and selectivity against a closely related kinase, PKA.     

Synthesis and use of sulfonamide-, sulfoxide-, or sulfone-containing aminoglycoside-CoA bisubstrates as mechanistic probes for aminoglycoside N-6'-acetyltransferase

Aminoglycoside-coenzyme A conjugates are challenging synthetic targets because of the wealth of functional groups and high polarity of the starting materials. We previously reported a one-pot synthesis of amide-linked aminoglycoside-CoA bisubstrates. These molecules are nanomolar inhibitors of aminoglycoside N-6'-acetyltransferase Ii (AAC(6')-Ii), an important enzyme involved in bacterial resistance to aminoglycoside antibiotics. We report here the synthesis and biological activity of five new aminoglycoside-CoA bisubstrates containing sulfonamide, sulfoxide, or sulfone groups. Interestingly, the sulfonamide-linked bisubstrate, which was expected to best mimic the tetrahedral intermediate, does not show improved inhibition when compared with amide-linked bisubstrates. On the other hand, most of the sulfone- and sulfoxide-containing bisubstrates prepared are nanomolar inhibitors of AAC(6')-Ii.