Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mimetics prepared by ring-closing metathesis of C-terminal allylglycines with an N-terminal beta-vinyl-substituted phosphotyrosyl mimetic

Preferential binding of ligands to Grb2 SH2 domains in beta-bend conformations has made peptide cyclization a logical means of effecting affinity enhancement. This is based on the concept that constraint of open-chain sequences to bend geometries may reduce entropy penalties of binding. The current study extends this approach by undertaking ring-closing metathesis (RCM) macrocyclization between i and i+3 residues through a process involving allylglycines and beta-vinyl-functionalized residues. Ring closure in this fashion results in minimal macrocyclic tetrapeptide mimetics. The predominant effects of such macrocyclization on Grb2 SH2 domain binding affinity were increases in rates of association (from 7- to 16-fold) relative to an open-chain congener, while decreases in dissociation rates were less pronounced (approximately 2-fold). The significant increases in association rates were consistent with pre-ordering of solution conformations to near those required for binding. Data from NMR experiments and molecular modeling simulations were used to interpret the binding results. An understanding of the conformational consequences of such i to i+3 ring closure may facilitate its application to other systems where bend geometries are desired.     

The discovery of potent inhibitors of aldosterone synthase that exhibit selectivity over 11-β-hydroxylase

Aldosterone, the final component of the renin–angiotensin–aldosterone system, plays an important role in the pathophysiology of hypertension and congestive heart failure. Aldosterone synthase (CYP11B2) catalyzes the last three steps of aldosterone biosynthesis, and as such appears to be a target for the treatment of these disorders. A sulfonamide–imidazole scaffold has proven to be a potent inhibitor of CYP11B2. Furthermore, this scaffold can achieve high levels of selectivity for CYP11B2 over CYP11B1, a key enzyme in the biosynthesis of cortisol.     

Aqueous Extract of the Pericarp of Sapindus trifoliatus Fruits: A Novel ‘Green’ Catalyst for the Aldimine Synthesis

The catalytic efficiency in organic synthesis of the aqueous extract of the pericarp of Sapindus trifoliatus fruits was evaluated. The synthesis of a series of aldimines from aromatic aldehydes and amines was successfully catalyzed by the extract, whereas aromatic ketones and amines did not yield ketimines under comparable reaction conditions, indicating the chemoselective catalysis of the extract. The catalytic activity of the extract is due to saponins, which have a common structural skeleton containing a pentacyclic triterpenoid part substituted with different carbohydrate side chains. The mild conditions, high yields, and short reaction times not only make this protocol a valuable alternative to the conventional methods, but it also becomes significant under the roof of environmentally greener and safer processes.     

Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor

HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.     

Utilization of a nitrobenzoxadiazole (NBD) fluorophore in the design of a Grb2 SH2 domain-binding peptide mimetic

Fluorescence labeling has become a general technique for studying the intracellular accumulation and localization of exogenously administered materials. Reported herein is a low nanomolar affinity Grb2 SH2 domain-binding antagonist that utilizes the environmentally-sensitive nitrobenzoxadiazole (NBD) fluorophore as a naphthyl replacement. This novel agent should serve as a useful tool to visualize the actions of this class of Grb2 SH2 domain-binding antagonists in whole cell systems.     

Virtual screening application of a model of full-length HIV-1 integrase complexed with viral DNA

To address the absence of experimental data on the full-length structure of HIV-1 integrase and the way it binds to viral and human DNA, we had previously [Karki, R. G.; Tang, Y.; Burke, T. R., Jr.; Nicklaus, M. C. J. Comput. Aided Mol. Des.2004, 18, 739] constructed models of full-length HIV-1 integrase complexed with models of viral and human DNA. Here we describe the discovery of novel HIV-1 integrase strand transfer inhibitors based on one of these models. Virtual screening methods including docking and filtering by predicted ADME/Tox properties yielded several microM level inhibitors of the strand transfer reaction catalyzed by wild-type HIV-1 integrase.     

Application of azide-alkyne cycloaddition 'click chemistry' for the synthesis of Grb2 SH2 domain-binding macrocycles

Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing macrocycles based on the Grb2 SH2 domain-binding motif, 'Pmp-Ac(6)c-Asn', where Pmp and Ac(6)c stand for 4-phosphonomethylphenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1mM substrate concentrations, cyclization of monomeric units occurred. At 2mM substrate concentrations the predominant products were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a K(d) value of 0.23microM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of 'click chemistry' to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.     

Azido-containing aryl beta-diketo acid HIV-1 integrase inhibitors

Aryl beta-diketo acids (ADK) comprise a general class of potent HIV-1 integrase (IN) inhibitors, which can exhibit selective inhibition of strand transfer reactions in extracellular recombinant IN assays and provide potent antiviral effects in HIV-infected cells. Recent studies have shown that polycyclic aryl or aryl rings bearing aryl-containing substituents are components of potent members of this class. Reported herein is the first use of azido functionality as an aryl replacement in beta-diketo acid IN inhibitors. The ability of azido-containing inhibitors to exhibit potent inhibition of IN and antiviral protection in HIV-infected cells, renders the azide group of potential value in the further development of ADK-based IN inhibitors.     

Three-dimensional quantitative structure–activity relationship (3D-QSAR) of 3-aryloxazolidin-2-one antibacterials

Three-dimensional quantitative structure–activity relationship (3D-QSAR) studies for 3-aryloxazolidin-2-one antibacterials were performed using the genetic function approximation algorithm. This study was performed using 60 compounds, in which the QSAR models were developed using a training set of 50 compounds. The in vitro minimum inhibitory concentration (MIC) against Staphylococcus aureus SFCO-1a was used for the study. The predictive ability of the QSAR model was evaluated by using a test set of 10 compounds. The statistical quality of the QSAR models was assessed using statistical parameters r², r²_cv (cross-validated r²), r²_pred (predictive r²) and lack of fit measure (LOF). The results obtained indicate that the antibacterial activity of the 3-aryloxazolidin-2-ones is strongly dependent on electronic factor as expressed by lowest unoccupied molecular orbital energy (LUMO), spatial factor as expressed by density and thermodynamic factors accounted for by molar refractivity and heat of formation. The model is presently being used to design and predict new potent molecules prior to synthesis.     

Utilization of a common pathway for the synthesis of high affinity macrocyclic Grb2 SH2 domain-binding peptide mimetics that differ in the configuration at one ring junction

As typified by 2-{(9S,10S,14R,18S)-18-(2-amino-2-oxoethyl)-14-[(5-methyl-1H-indol-1-yl)methyl]-8,17,20-trioxo-10-[4-(phosphonomethyl)phenyl]-7,16,19-triazaspiro[5.14]icos-11-en-9-yl}acetic acid ((14R)-1b), ring-closing methathesis-derived macrocyclic tetrapeptide mimetics have recently been reported that bind with high affinity to Grb2 SH2 domains in both extracellular and whole-cell assays. The synthetic complexity of this class of agents limits further therapeutic development. Although a significant component of this synthetic complexity arises from the presence of three stereogenic centers, C(9) (S), C(10) (S), and C(14) (R), it is unclear whether stereoselective introduction of defined configuration at C(14) is required for high-affinity binding. Reported herein is a synthetic route to these macrocycles lacking stereoselectivity in the formation of the C(14) ring junction, which is four synthetic steps shorter than the original stereoselective synthesis. Separation of C(14)-epimers obtained by this approach was achieved by preparative HPLC. Molecular-dynamics studies of ligands bound to the Grb2 SH2 domain protein indicated that the (14R)-configuration should display more-favorable interactions with the protein relative to the (14S)-epimer. Indeed, although surface-plasmon-resonance-derived binding constants to Grb2 SH2 domain protein indicated that the affinity of the (14R)-epimer (KD = 4.8 nM) is greater than that of the (14S)-epimer (KD = 11 nM), it is only marginally so. Therefore, little affinity would be lost through a non-stereoselective synthesis of the C(14)-center. Further studies are in progress to explore reduced structural complexity at the C(14)-center.