Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein-Protein Interaction between CTLA-4 and B7-1.

Currently, various pharmaceutical modalities are being developed rapidly. Targeting protein-protein interactions (PPIs) is an important objective in such development. Cyclic peptides, because they have good specificity and activity, have been attracting much attention as an alternative to antibody drugs. However, cyclic peptides involve some difficulties, such as oral availability and cell permeability. Therefore, while small-molecule drugs still present many benefits, the screening of functional small-molecule compounds targeting PPIs requires a great deal of time and effort, including structural analysis of targets and hits. In this study, we investigated a rational two-step strategy to design small-molecule compounds targeting PPIs. First, we obtained inhibitory cyclic peptides that bind to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) by ribosomal display using PUREfrex® (PUREfrex®RD) to get structure-activity relation (SAR) information. Based on that information, we converted cyclic peptides to small molecules using PepMetics® scaffolds that can mimic the α-helix or ß-turn of the peptide. Finally, we succeeded in generating small-molecule compounds with good IC50 (single-digit µM values) against CTLA-4. This strategy is expected to be a useful approach for small-molecule design targeting PPIs, even without having structural information such as that associated with X-ray crystal structures.

TP0586532, a Novel Non-Hydroxamate LpxC Inhibitor: Potentiating Effect on In Vitro Activity of Meropenem against Carbapenem-Resistant Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat to public health requiring the development of novel therapies. TP0586532 is a novel non-hydroxamate LpxC inhibitor that inhibits the synthesis of lipopolysaccharides, which are components of the outer membranes of Gram-negative bacteria. Based on the mechanism of action of TP0586532, we hypothesized that it might enhance the antibacterial activity of other antibiotics by increasing the permeability of the outer bacterial membrane. The combination of TP0586532 with meropenem, amikacin, cefepime, piperacillin, and tigecycline showed synergistic and additive effects against carbapenem-susceptible Klebsiella pneumoniae and Escherichia coli. Checkerboard experiments against 21 carbapenem-resistant K. pneumoniae and E. coli strains (13 blaKPC+, 5 blaNDM-1+, 2 blaVIM+, and 1 blaIMP+) showed that the combination of TP0586532 with meropenem yielded synergistic and additive effects against 9 and 12 strains, respectively. In a time-kill assay examining 12 CRE strains, synergistic effects were observed when TP0586532 was combined with meropenem against many of the strains. A membrane permeability assay using ethidium bromide (EtBr) was performed to investigate the mechanism of the potentiating effect. TP0586532 increased the influx of EtBr into a CRE strain, suggesting that TP0586532 increased membrane permeability and facilitated intracellular access for the antibiotics. Our study demonstrates that TP0586532 potentiates the in vitro antibacterial activity of meropenem against CRE. Combination therapy consisting of TP0586532 and meropenem has potential as a treatment for CRE infections. IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent public health threat, as therapeutic options are limited. TP0586532 is a novel LpxC inhibitor that inhibits the synthesis of lipopolysaccharides in the outer membranes of Gram-negative bacteria. Here, we demonstrated the potentiating effects of TP0586532 on the antibacterial activity of meropenem against CRE harboring various types of carbapenemase genes (blaKPC+, blaNDM-1+ blaVIM+, and blaIMP+). TP0586532 also augmented the bactericidal effects of meropenem against CRE strains, even against those with a high level of resistance to meropenem. The potentiating effects were suggested to be mediated by an increase in bacterial membrane permeability. Our study revealed that a combination therapy consisting of TP0586532 and meropenem has the potential to be a novel therapeutic option for CRE infections.

Pharmacodynamic target assessment and prediction of clinically effective dosing regimen of TP0586532, a novel non-hydroxamate LpxC inhibitor, using a murine lung infection model.

INTRODUCTION: TP0586532 is a novel non-hydroxamate UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) inhibitor. Pharmacokinetic/pharmacodynamic (PK/PD) indices and magnitude of index that correlated with the efficacy of TP0586532 were determined and used to estimate the clinically effective doses of TP0586532. METHODS: Dose-fractionation studies were conducted using a murine neutropenic lung infection model caused by carbapenem-resistant Enterobacteriaceae. The relationships between the efficacy and the PK/PD index (the maximum unbound plasma concentration divided by the MIC [fCmax/MIC], the area under the unbound plasma concentration-time curve from 0 to 24 h divided by the MIC, and the cumulative percentage of a 24-h period that the unbound plasma concentration exceeds the MIC) were determined using an inhibitory sigmoid maximum-effect model. In addition, the magnitudes of fCmax/MIC were evaluated using the dose-response relationships for each of the seven carbapenem-resistant strains of Enterobacteriaceae. Furthermore, the clinically effective doses of TP0586532 were estimated using the predicted human PK parameters, the geometric mean of fCmax/MIC, and the MIC90 for carbapenem-resistant Klebsiella pneumoniae. RESULTS: The PK/PD index that best correlated with the efficacy was the fCmax/MIC. The geometric means of the fCmax/MIC associated with the net stasis and 1-log reduction endpoints were 2.30 and 3.28, respectively. The clinically effective doses of TP0586532 were estimated to be 1.24-2.74 g/day. CONCLUSION: These results indicate the potential for TP0586532 to have clinical efficacy at reasonable doses against infections caused by carbapenem-resistant Enterobacteriaceae. This study provided helpful information for a clinically effective dosing regimen of TP0586532.

TP0586532, a non-hydroxamate LpxC inhibitor, reduces LPS release and IL-6 production both in vitro and in vivo.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an essential enzyme in the biosynthesis of Lipid A, an active component of lipopolysaccharide (LPS), from UDP-3-O-acyl-N-acetylglicosamine. LPS is a major component of the cell surface of Gram-negative bacteria. LPS is known to be one of causative factors of sepsis and has been associated with high mortality in septic shock. TP0586532 is a novel non-hydroxamate LpxC enzyme inhibitor. In this study, we examined the inhibitory effect of TP0586532 on the LPS release from Klebsiella pneumoniae both in vitro and in vivo. Our results confirmed the inhibitory effect of TP0586532 on LPS release from the pathogenic bacterial species. On the other hand, meropenem and ciprofloxacin increase the level of LPS release. Furthermore, the effects of TP0586532 on LPS release and interleukin (IL)-6 production in the lung were determined using a murine model of pneumonia caused by K. pneumoniae. As observed in the in vitro study, TP0586532 showed the marked inhibitory effect on LPS release in the lungs, whereas meropenem- and ciprofloxacin-treated mice showed higher levels of LPS release and IL-6 production in the lungs as compared to those in the lungs of vehicle-treated mice. Moreover, TP0586532 used in combination with meropenem and ciprofloxacin attenuated the LPS release and IL-6 production induced by meropenem and ciprofloxacin in the lung. These results indicate that the inhibitory effect of TP0586532 on LPS release from pathogenic bacteria might be of benefit in patients with sepsis.

TP0586532, a non-hydroxamate LpxC inhibitor, has in vitro and in vivo antibacterial activities against Enterobacteriaceae.

The emergence of multi-drug resistant pathogenic bacteria, especially Gram-negative bacteria, is a worldwide health problem. New antibiotics directed at previously unexplored targets are urgently needed to overcome resistance to existing antibiotic classes. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an attractive target for a new antibacterial agent. Although a number of LpxC inhibitors have been identified, none have been approved as antibacterial agents. These LpxC inhibitors contain a hydroxamate moiety, which is a robust zinc ion chelator. The nonspecific inhibition of metalloenzymes through zinc ion chelation is one of possibilities leading to unwanted side effects. Herein, we report that TP0586532, a non-hydroxamate LpxC inhibitor, has a broad spectrum of antibacterial activity against carbapenem-resistant Enterobacteriaceae. The MIC90 of TP0586532 against clinical isolates of carbapenem-resistant Klebsiella pneumoniae was 4 µg ml-1. TP0586532 also showed an in vivo efficacy against murine systemic, urinary tract and lung infection models caused by meropenem- or ciprofloxacin-resistant strains. The estimated maximum unbound plasma concentration value at the effective dose of TP0586532 in murine infection models was around 13 µg ml-1. TP0586532 is predicted to exhibit a in vivo efficacy without cardiovascular toxicity and showed the potential of non-hydroxamate LpxC inhibitors as antibacterial agents against carbapenem-resistant Enterobacteriaceae.

Visualized and Quantitative Conformational Analysis of Peptidomimetics.

Protein-protein interactions (PPIs) are fundamentally important and challenging drug targets. Peptidomimetic molecules of various types have been developed to modulate PPIs. A particularly promising drug discovery strategy, structural peptidomimetics, was designed based on special mimicking of side-chain Cα-Cß bonds. It is simple and versatile. Nevertheless, no quantitative method has been established to evaluate its similarity to a target peptide motif. We developed two methods that enable visual, comprehensive, and quantitative analysis of peptidomimetics: peptide conformation distribution (PCD) plot and peptidomimetic analysis (PMA) map. These methods specifically examine multiple side-chain Cα-Cß bonds of a peptide fragment motif and their corresponding bonds (pseudo-Cα-Cß bonds) in a mimetic molecule instead of φ and ψ angles of a single amino acid in the traditional Ramachandran plot. The PCD plot is an alignment-free method, whereas the PMA map is an alignment-based method providing distinctive and complementary analysis. Results obtained from analysis using these two methods indicate our multifacial α-helix mimetic scaffold 12 as an excellent peptidomimetic that can precisely mimic the spatial positioning of side-chain functional groups of α-helix. These methods are useful for visualized and quantified evaluation of peptidomimetics and for the rational design of new mimetic scaffolds.

Generalized-ensemble method study: A helix-mimetic compound inhibits protein-protein interaction by long-range and short-range intermolecular interactions.

A heterocyclic compound mS-11 is a helix-mimetic designed to inhibit binding of an intrinsic disordered protein neural restrictive silence factor/repressor element 1 silencing factor (NRSF/REST) to a receptor protein mSin3B. We apply a generalized ensemble method, multi-dimensional virtual-system coupled molecular dynamics developed by ourselves recently, to a system consisting of mS-11 and mSin3B, and obtain a thermally equilibrated distribution, which is comprised of the bound and unbound states extensively. The lowest free-energy position of mS-11 coincides with the NRSF/REST position in the experimentally-determined NRSF/REST-mSin3B complex. Importantly, the molecular orientation of mS-11 is ordering in a wide region around mSin3B. The resultant binding scenario is: When mS-11 is distant from the binding site of mSin3B, mS-11 descends the free-energy slope toward the binding site maintaining the molecular orientation to be advantageous for binding. Then, finally a long and flexible hydrophobic sidechain of mS-11 fits into the binding site, which is the lowest-free-energy complex structure inhibiting NRSF/REST binding to mSin3B.

Lead optimization of 2-hydroxymethyl imidazoles as non-hydroxamate LpxC inhibitors: Discovery of TP0586532.

Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the development of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with existing drugs has been earnestly desired. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a drug target that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result, a development candidate, TP0586532, was created that is effective against carbapenem-resistant Klebsiella pneumoniae and does not pose a cardiovascular risk.

Fragment-Based Discovery of Novel Non-Hydroxamate LpxC Inhibitors with Antibacterial Activity.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation. A series was evolved from a fragment where a glycine moiety complexes zinc, which achieved low nanomolar potency in an enzyme functional assay but poor antibacterial activity on cell cultures. A second series was based on a fragment that chelated zinc through an imidazole moiety. Structure-guided design led to a 2-(1S-hydroxyethyl)-imidazole derivative exhibiting low nanomolar inhibition of LpxC and a minimum inhibitory concentration (MIC) of 4 µg/mL against Pseudomonas aeruginosa, which is little affected by the presence of albumin.

Ursolic acid, an antagonist for transforming growth factor (TGF)-beta1.

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine which is involved in extracellular matrix modulation, has a major role in the pathogenesis and progression of fibrotic diseases. We now report the effects of ursolic acid on TGF-beta1 receptor binding and TGF-beta1-induced cellular functions in vitro. Ursolic acid inhibited [(125)I]-TGF-beta1 receptor binding to Balb/c 3T3 mouse fibroblasts with an IC(50) value of 6.9+/-0.8 microM. Ursolic acid dose-dependently recovered reduced proliferation of Minc Mv1Lu cells in the presence of 5 nM of TGF-beta1 and attenuated TGF-beta1-induced collagen synthesis and production in human fibroblasts. Molecular dynamics simulations suggest that ursolic acid may interact with the hydrophobic region of the dimeric interface and thereby inhibit the binding of TGF-beta1 to its receptor. All these findings taken together show that ursolic acid functions as an antagonist for TGF-beta1. This is the first report to show that a small molecule can inhibit TGF-beta1 receptor binding and influence functions of TGF-beta1.

A novel low molecular weight antagonist of vascular endothelial growth factor receptor binding: VGA1155.

Vascular endothelial growth factor (VEGF) plays a pivotal role in the processes of angiogenesis, which is essential for the growth of solid tumors and their metastasis. Because VEGF is a critical factor in tumor survival, inhibiting VEGF would provide significant benefits in tumor therapy. To identify a compound that inhibits the binding of VEGF to its receptor, we used a high throughput screening method, finding that small molecular compounds inhibited VEGF binding. Among active compounds, 5-[N-methyl-N-(4-octadecyloxyphenyl)acetyl]amino-2-methylthiobenzoic acid (VGA1155) was selected for its potent inhibition of binding. VGA1155 inhibited [(125)I] VEGF binding to two cell lines, NIH3T3-fms-like tyrosine kinase-1 (VEGF receptor 1 transfected) cells and NIH3T3-kinase insert domain containing receptor/fetal liver kinase-1 (KDR/Flk-1; VEGF receptor 2 transfected), in a concentration-dependent manner. VGA1155 did not inhibit the binding of several other growth factors or cytokines to their receptors. Based on the results of surface plasmon resonance analysis using Biacore S51 system, it appears that this binding inhibitory property may be based on the association of VGA1155 with VEGF receptor 2 (KDR/Flk-1). Further, the interference in VEGF binding by VGA1155 in turn induces the inhibition of VEGF-induced KDR/Flk-1 autophosphorylation. VGA1155 also reduced intradermal VEGF-induced vascular permeability in guinea pigs. These findings indicate that VGA1155 inhibits not only VEGF binding to its receptors through association with KDR/Flk-1 but also VEGF function in vivo. These VGA1155 activities may provide a useful basis for the development of antiangiogenic and antitumor agents.

Structural consideration of macrolide antibiotics in relation to the ribosomal interaction and drug design.

Macrolide antibiotics exert antimicrobial effects by binding to the peptidyl transferase center of the 50S subunit of bacterial ribosomes and inhibiting protein synthesis. Hence, the structure of macrolides and their interaction with bacterial ribosomes have been investigated in order to understand the structural mechanisms of macrolide-ribosome interaction. Most macrolides have been found tom adopt a common conformation both in crystal form and in solution, which is believed to play an important role for binding to bacterial ribosomes as well as representing bi-facial property essential for excellent biological functions of macrolides. Chemical footprinting and mutant analysis have offered topological information on macrolide-ribosome interaction at the nucleotide level. Recently, crystal structures of the 50S ribosomal subunit and the 50S subunit with macrolide antibiotics have been published. These crystal structures provide much structural information on macrolide-ribosome interaction at the atomic level and will enable structure-based drug design of novel macrolide antibiotics with potent activity against resistant strains.

A novel parallel algorithm for large-scale Fock matrix construction with small locally distributed memory architectures: RT parallel algorithm.

We developed a novel parallel algorithm for large-scale Fock matrix calculation with small locally distributed memory architectures, and named it the "RT parallel algorithm." The RT parallel algorithm actively involves the concept of integral screening, which is indispensable for reduction of computing times with large-scale biological molecules. The primary characteristic of this algorithm is parallel efficiency, which is achieved by well-balanced reduction of both communicating and computing volume. Only the density matrix data necessary for Fock matrix calculations are communicated, and the data once communicated are reutilized for calculations as many times as possible. The RT parallel algorithm is a scalable method because required memory volume does not depend on the number of basis functions. This algorithm automatically includes a partial summing technique that is indispensable for maintaining computing accuracy, and can also include some conventional methods to reduce calculation times. In our analysis, the RT parallel algorithm had better performance than other methods for massively parallel processors. The RT parallel algorithm is most suitable for massively parallel and distributed Fock matrix calculations for large-scale biological molecules with more than thousands of basis functions.