Synthesis of potent and highly selective nonguanidine azetidinone inhibitors of human tryptase

Azetidinones such as BMS-363131 (2) and BMS-363130 (3), which contain a guanidine group in the C-3 side chain were previously shown to be very potent inhibitors of human tryptase with high selectivity versus other serine proteases, including trypsin. In this letter, we describe the discovery of a number of potent azetidinone tryptase inhibitors in which the guanidine moiety at the ring C-3 position is replaced with primary or secondary amine or aminopyridine functionality. In particular, BMS-354326 (4) is a highly potent tryptase inhibitor (IC(50)=1.8 nM), which has excellent selectivity against trypsin and most other related serine proteases.     

Investigation of mechanism-based thrombin inhibitors: Implications of a highly conserved water molecule for the binding of coumarins within the S pocket

The synthesis of novel coumarins bearing on the lateral side chain in the 3-position an amine or a guanidine group is described. In vitro evaluation highlighted 14d which possesses a meta aniline side chain as a very potent THR inhibitor. Surprisingly, the introduction of a guanidine moiety always led to a decrease in THR inhibiting properties. We, thus, used docking experiments to rationalize the SAR in the series. This study showed the crucial role of a conserved water molecule in the specificity pocket of THR during docking simulation in order to explain the inactivity of guanidine derivatives.     

Peptidyl aldehydes as reversible covalent inhibitors of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. PTP inhibitors provide potential treatment of human diseases/conditions such as diabetes and obesity as well as useful tools for studying the function of PTPs in signaling pathways. In this work, we have shown that certain aryl-substituted aldehydes act as reversible, slow-binding inhibitors of modest potency against PTP1B, SHP-1, and a dual-specificity phosphatase, VHR. Attachment of the tripeptide Gly-Glu-Glu to the para position of cinnamaldehyde resulted in an inhibitor (Cinn-GEE) of substantially increased potency against all three enzymes (e.g., K(I) = 5.4 microM against PTP1B). The mechanism of inhibition was investigated using Cinn-GEE specifically labeled with (13)C at the aldehyde carbon and (1)H-(13)C heteronuclear single-quantum coherence spectroscopy. While Cinn-GEE alone showed a single cross-peak at delta 9.64 ((1)H) and delta 201 ((13)C), the PTP1B/Cinn-GEE complex showed three distinct cross-peaks at delta 7.6-7.8 ((1)H) and 130-137 ((13)C). Mutation of the catalytic cysteine (Cys-215 in PTP1B) into alanine had no effect on the cross-peaks, whereas mutation of a conserved active-site arginine (Arg-221 in PTP1B) to alanine abolished all three cross-peaks. Similar experiments with Cinn-GEE that had been labeled with (13)C at the benzylic position revealed a change in the hybridization state (from sp(2) to sp(3)) for the benzylic carbon as a result of binding to PTP1B. These results rule out the possibility of a free aldehyde, aldehyde hydrate, or hemithioacetal as the enzyme-bound inhibitor form. Instead, the data are consistent with the formation of an enamine between the aldehyde group of the inhibitor and the guanidine group of Arg-221 in the PTP1B active site. These aldehydes may provide a general core structure that can be further developed into highly potent and specific PTP inhibitors.     

Design of potent substrate-analogue inhibitors of canine renin.

Through a systematic study of structure-activity relationships, we designed potent renin inhibitors for use in dog models. In assays against dog plasma renin at neutral pH, we found that, as in previous studies of rat renin inhibitors, the structure at the P2 position appears to be important for potency. The substitution of Val for His at this position increases potency by one order of magnitude. At the P3 position, potency appears to depend on a hydrophobic side chain that does not necessarily have to be aromatic. Our results also support the approach of optimizing potency in a renin inhibitor by introducing a moiety that promotes aqueous solubility (an amino group) at the C-terminus of the substrate analogue. In the design of potent dog plasma renin inhibitors, the influence of the transition-state residue 4(S)-amino-3(S)-hydroxy-5-cyclohexylpentanoic acid (ACHPA)-commonly used as a substitute for the scissile-bond dipeptide to boost potency-is not obvious, and appears to be sequence dependent. The canine renin inhibitor Ac-paF-Pro-Phe-Val-statine-Leu-Phe-paF-NH2 (compound 15; IC50 of 1.7 nM against dog plasma renin at pH 7.4; statine, 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid; paF, para-aminophenylalanine) had a potent hypotensive effect when infused intravenously into conscious, sodium-depleted, normotensive dogs. Also, compound 15 concurrently inhibited plasma renin activity and had a profound diuretic effect.     

Novel non-active site inhibitor of Cryptosporidium hominis TS-DHFR identified by a virtual screen

The essential enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a validated drug target for many pathogens, but has been elusive in Cryptosporidium hominis, as active site inhibitors of the enzymes from related parasitic protozoa show decreased potency and lack of species specificity over the human enzymes. As a rational approach to discover novel inhibitors, we conducted a virtual screen of a non-active site pocket in the DHFR linker region. From this screen, we have identified and characterized a noncompetitive inhibitor, flavin mononucleotide (FMN), with micromolar potency that is selective for ChTS-DHFR versus the human enzymes. These results describe a novel allosteric pocket amenable to inhibitor targeting, and a lead compound with which to move towards potent, selective inhibitors of ChTS-DHFR.     

2-difluoromethyloestrone 3-O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).     

Discovery of potent PTP1B inhibitors via structure-based drug design,synthesis and in vitro bioassay of Norathyriol derivatives

Protein tyrosine phosphatase 1B (PTP1B) has recently been identified as a potential target of Norathyriol. Unfortunately, Norathyriol is not a potent PTP1B inhibitor, which somewhat hinders its further application. Based on the fact that no study on the relationship of chemical structure and PTP1B inhibitory activity of Norathyriol has been reported so far, we attempted to perform structural optimization so as to improve the potency for PTP1B. Via structure-based drug design (SBDD), a rational strategy based on the binding mode of Norathyriol to PTP1B, we designed 26 derivatives with substitutions at the four phenolic hydroxyl groups of Norathyriol. By chemical synthesis and in vitro bioassay, we identified seven PTP1B inhibitors that were more potent than Norathyriol, of which XWJ24 showed the highest potency (IC₅₀: 0.6 μM). We also found out that XWJ24 was a competitive inhibitor and showed the 4.5-fold selectivity over its close homolog, TC-PTP. Through molecular docking of XWJ24 against PTP1B, we highlighted the essential role of its hydrogen bond with Asp181 for PTP1B inhibition and identified a potential halogen bond with Asp48 that was not observed for Norathyriol. The current data indicate that our SBDD strategy is effective to discover potent PTP1B-targeted Norathyriol derivatives, and XWJ24 is a promising lead compound for further development.     

SAR studies of 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

Various 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one 1 derivatives have been synthesized and tested as inhibitors of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from pig liver. The inhibition results indicated that well-positioned dicarbonyl groups as well as the cyclopropyl group of 1 were essential for potent inhibition. Substitution at the 2-position of the ring system has a significant effect on inhibitor potency, while the 5-position can undergo substantial variations and retain inhibitor potency. In the compounds examined, 2-chloro substituted 12 is the best inhibitor of all with IC(50) of 15 nM, the rest of the synthesized analogues were less potent inhibitors than the parent compound.     

Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives

MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14 mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The mrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were cross-resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.     

Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.     

Use of intravenous peramivir for treatment of severe influenza A(H1N1)pdm09

Oral antiviral agents to treat influenza are challenging to administer in the intensive care unit (ICU). We describe 57 critically ill patients treated with the investigational intravenous neuraminidase inhibitor drug peramivir for influenza A (H1N1)pdm09 [pH1N1]. Most received late peramivir treatment following clinical deterioration in the ICU on enterically-administered oseltamivir therapy. The median age was 40 years (range 5 months-81 years). Common clinical complications included pneumonia or acute respiratory distress syndrome requiring mechanical ventilation (54; 95%), sepsis requiring vasopressor support (34/53; 64%), acute renal failure requiring hemodialysis (19/53; 36%) and secondary bacterial infection (14; 25%). Over half (29; 51%) died. When comparing the 57 peramivir-treated cases with 1627 critically ill cases who did not receive peramivir, peramivir recipients were more likely to be diagnosed with pneumonia/acute respiratory distress syndrome (p?=?0.0002) or sepsis (p?=?<0.0001), require mechanical ventilation (p?=?<0.0001) or die (p?=?<0.0001). The high mortality could be due to the pre-existing clinical severity of cases prior to request for peramivir, but also raises questions about peramivir safety and effectiveness in hospitalized and critically ill patients. The use of peramivir merits further study in randomized controlled trials, or by use of methods such as propensity scoring and matching, to assess clinical effectiveness and safety.     

Inhibition of Vesicular Glutamate Uptake by Rose Bengal-Related Compounds: Structure–Activity Relationship

Synaptic vesicular accumulation of glutamate is a vital initial step in glutamate transmission. We have previously shown that Rose Bengal, a polyhalogenated fluorescein analog, is a potent inhibitor of glutamate uptake into synaptic vesicles. Here, we report the structural features of Rose Bengal required for this inhibition. Various Rose Bengal-related compounds, with systematic structural variations, were tested. Results indicate that the four iodo groups and the phenyl group attached to the xanthene moiety are critical for potent inhibitory activity. Replacement of these groups with two iodo groups and an alkyl group, respectively, results in substantial reduction in potency. Of further interest in creating high potency is the critical nature of the oxygen atom which links the two benzene rings of xanthene. Thus, the phenyl group and multiple iodo groups, as well as the bridging oxygen of xanthene, are crucial elements of Rose Bengal required for its potent inhibitory action.     

Structure-Activity Relationships of Benzbromarone Metabolites and Derivatives as EYA Inhibitory Anti-Angiogenic Agents

The tyrosine phosphatase activity of the phosphatase-transactivator protein Eyes Absent (EYA) is angiogenic through its roles in endothelial cell migration and tube formation. Benzbromarone, a known anti-gout agent, was previously identified as an inhibitor of EYA with anti-angiogenic properties. Here we show that the major metabolite of BBR, 6-hydroxy benzbromarone, is a significantly more potent inhibitor of cell migration, tubulogenesis and angiogenic sprouting. In contrast, other postulated metabolites of BBR such as 5-hydroxy benzbromaorne and 1’-hydroxy benzbromarone are less potent inhibitors of EYA tyrosine phosphatase activity as well as being less effective in cellular assays for endothelial cell migration and angiogenesis. Longer substituents at the 2 position of the benzofuran ring promoted EYA3 binding and inhibition, but were less effective in cellular assays, likely reflecting non-specific protein binding and a resulting reduction in free, bio-available inhibitor. The observed potency of 6-hydroxy benzbromarone is relevant in the context of the potential re-purposing of benzbromarone and its derivatives as anti-angiogenic agents. 6-hydroxy benzbromarone represents a metabolite with a longer half-life and greater pharmacological potency than the parent compound, suggesting that biotransformation of benzbromarone could contribute to its therapeutic activity.     

Mechanism of inhibition of the GluR2 AMPA receptor channel opening by 2,3-benzodiazepine derivatives

2,3-Benzodiazepine derivatives are drug candidates synthesized for potential treatment of various neurodegenerative diseases involving the excessive activity of AMPA receptors. Here we describe a rapid kinetic investigation of the mechanism of inhibition of the GluR2Qflip AMPA receptor channel opening by two 2,3-benzodiazepine derivatives that are structurally similar (BDZ-2 and BDZ-3). Using a laser-pulse photolysis technique with a time resolution of approximately 60 mus, we measured the effects of these inhibitors on both the channel opening rate and the whole-cell current amplitude. We found that both compounds preferably inhibit the open-channel state, although BDZ-2 is a more potent inhibitor in that it inhibits the open-channel state approximately 5-fold stronger than BDZ-3 does. Both compounds bind to the same noncompetitive site. Binding of an inhibitor to the receptor involves the formation of a loose, partially conducting channel intermediate, which rapidly isomerizes to a tighter complex. The isomerization reaction is identified as the main step at which the receptor distinguishes the structural difference between the two compounds. These results suggest that addition of a bulky group at the N-3 position on the diazepine ring, as in BDZ-3, does not alter the mechanism of action, or the site of binding, but does lower the inhibitory potency, possibly due to an unfavorable interaction of a bulky group at the N-3 position with the receptor site. The new mechanistic revelation about the structure-reactivity relationship is useful in designing conformation-specific, more potent noncompetitive inhibitors for the GluR2 AMPA receptor.     

Synthesis and biological evaluation of histone deacetylase inhibitors that are based on FR235222: a cyclic tetrapeptide scaffold

We outline the synthesis of six novel derivatives that are based on a recently discovered HDAC inhibitor FR235222. Our work is the first report utilizing a novel binding element, guanidine, as metal coordinators in HDAC inhibitors. Further, we demonstrate that these compounds show cytotoxicity that parallels their ability to inhibit deacetylase activity, and that the most potent compounds maintain an L-Phe at position 1, and a D-Pro at position 4. Both inhibition of HDAC activity and cytotoxicity against the pancreatic cancer cell line BxPC3 are exhibited by these compounds, establishing that a guanidine unit can be utilized successfully to inhibit HDAC activity.     

Peptidyl aldehydes as reversible covalent inhibitors of SRC homology 2 domains

Src homology 2 (SH2) domains are phosphotyrosine- (pY-) binding modules found in a variety of signal-transducing proteins and constitute an important class of drug targets for the treatment of signaling related diseases/conditions. To date, a large number of peptidic as well as nonpeptidic SH2 domain inhibitors have been reported. However, all of these inhibitors contain a negatively charged pY mimetic as the core structure and generally have poor membrane permeability. We report here that peptidyl cinnamaldehydes function as reversible, slow-binding inhibitors toward the SH2 domains of protein tyrosine phosphatase SHP-1. Specific interactions between the SH2 domains and the aldehydes were assessed by their ability to relieve the autoinhibitory effect of the N-terminal SH2 domain on SHP-1 catalytic activity and the surface plasmon resonance technique. The most potent inhibitor (Cinn-GEE) displayed a K(D) value of 1.3 microM against the N-terminal SH2 domain of SHP-1. The mechanism of inhibition was investigated by site-directed mutagenesis and by using Cinn-GEE specifically labeled with (13)C at the aldehyde carbon and (1)H-(13)C heteronuclear single-quantum coherence spectroscopy. The proposed mechanism involves the formation of an initial noncovalent E.I complex, which is slowly converted into a covalent imine/enamine adduct (E.I) between the aldehyde group of the inhibitor and the guanidine group of Arg betaB5 in the pY-binding pocket of the SH2 domains. These aldehydes should provide a general, neutral pharmacophore for the further development of potent, specific, and membrane-permeable SH2 domain inhibitors.     

Amide-based derivatives of β-alanine hydroxamic acid as histone deacetylase inhibitors: Attenuation of potency through resonance effects

A library of amide-linked derivatives of β-alanine hydroxamic acid were prepared (2-7) and the activity as inhibitors of Zn(II)-containing histone deacetylases (HDACs) determined in vitro against HDAC1 and the anti-proliferative activity determined in BE(2)-C neuroblastoma cells. The IC(50) values of the best-performing compounds (3-7) against HDAC1 ranged between 38 and 84μM. The least potent compound (2) inhibited a maximum of only 40% HDAC1 activity at 250μM. The anti-proliferative activity of 2-7 at 50μM against BE(2)-C neuroblastoma cells ranged between 57.0% and 88.6%. The structural similarity between the potent HDAC inhibitor trichostatin A (TSA, 1; HDAC1, IC(50) 12nM) and the present compounds (2-7) was high at the Zn(II) coordinating hydroxamic acid head group; and in selected compounds (2, 5), at the 4-(dimethylamino)phenyl tail. The significantly reduced potency of 2-7 relative to 1 underscores the rank importance of the linker region as part of the HDAC inhibitor pharmacophore. Molecular modeling of 1-7 using HDAC8 as the template suggested that the conformationally constrained 4'-methyl group of 1 may contribute to HDAC inhibitor potency through a sandwich-like interaction with a hydrophobic region containing F152 and F208; and that the absence of this group in 2-7 may reduce potency. The close proximity of the 5'-carbonyl oxygen atom in 2-7 to the sulfur atom of Met274 in HDAC8 or the corresponding isobutyl group of Leu274 in HDAC1 may attenuate potency through repulsive steric and dipole-dipole forces. In a unique resonance stabilized form of 2, this interaction could manifest as stronger ion-dipole repulsive forces, resulting in a further decrease in potency. This work suggests that resonance structures of HDAC inhibitors could modulate intermolecular interactions with HDAC targets, and potency.     

In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2)

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group between nitrogen atoms (N(1) and N(2)) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhibition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450 isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In that context, 5 might be suitable for further drug development.     

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.     

Structure-activity relationships of rationally designed AMACR 1A inhibitors

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the ‘racemisation’ reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure–activity relationship study has been performed. This paper describes the first structure–activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC₅₀ = 400–750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure–activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.