Discovery of LYS006, a Potent and Highly Selective Inhibitor of Leukotriene A4 Hydrolase.

The cytosolic metalloenzyme leukotriene A4 hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

Identification of a 3-ß-homoalanine conjugate of brusatol with reduced toxicity in mice.

Brusatol, a quassinoid natural product, is effective against multiple diseases including hematologic malignancies, as we reported recently by targeting the PI3Kγ isoform, but toxicity limits its further development. Herein, we report the synthesis of a series of conjugates of brusatol with amino acids and short peptides at its enolic hydroxyl at C-3. A number of conjugates with smaller amino acids and peptides demonstrated activities comparable to brusatol. Through in vitro and in vivo evaluations, we identified UPB-26, a conjugate of brusatol with a L- ß-homoalanine, which exhibits good chemical stability at physiological pH's (SGF and SIF), moderate rate of conversion to brusatol in both human and rat plasmas, improved mouse liver microsomal stability, and most encouragingly, enhanced safety compared to brusatol in mice upon IP administration.

Synthesis and Inhibitory Studies of Phosphonic Acid Analogues of Homophenylalanine and Phenylalanine towards Alanyl Aminopeptidases.

A library of novel phosphonic acid analogues of homophenylalanine and phenylalanine, containing fluorine and bromine atoms in the phenyl ring, have been synthesized. Their inhibitory properties against two important alanine aminopeptidases, of human (hAPN, CD13) and porcine (pAPN) origin, were evaluated. Enzymatic studies and comparison with literature data indicated the higher inhibitory potential of the homophenylalanine over phenylalanine derivatives towards both enzymes. Their inhibition constants were in the submicromolar range for hAPN and the micromolar range for pAPN, with 1-amino-3-(3-fluorophenyl) propylphosphonic acid (compound 15c) being one of the best low-molecular inhibitors of both enzymes. To the best of our knowledge, P1 homophenylalanine analogues are the most active inhibitors of the APN among phosphonic and phosphinic derivatives described in the literature. Therefore, they constitute interesting building blocks for the further design of chemically more complex inhibitors. Based on molecular modeling simulations and SAR (structure-activity relationship) analysis, the optimal architecture of enzyme-inhibitor complexes for hAPN and pAPN were determined.

Surface charge-based rational design of aspartase modifies the optimal pH for efficient ß-aminobutyric acid production.

ß-Aminobutyric acid (BABA) can be widely used in the preparation of anti-tumor drugs, AIDS drugs, penicillin antibiotics, and plant initiators. However, the efficient, economical, and environmentally friendly production of BABA still faces challenges. Its important production enzyme, aspartase, catalyzes the substrate crotonic acid, and depends on harsh conditions, such as high temperatures and the presence of strong alkali. Here, we modified the surface charge of the enzyme to enable it to become more negatively charged (K19E, N87E, N125D, S133D, Q262E, and N451E; from -60 to -80), reducing its optimal pH from 9.0 to 8.0. The M20 enzyme showed improved specific activity (400.21 mU/mg at pH 8.0; 2.47-fold that of aspartase), and at pH 7.0, its activity increased 3-fold. The thermal stability of the enzyme was also improved. For the production of BABA, a 500 g/L whole-cell transformation was obtained with a 1.41-fold increase in yield, and the final production of BABA reached 556.1 g/L within 12 h. Our method provides a new strategy for modifying the characteristics of the enzyme through the modification of its surface charge, which also represents the first modification of the optimal pH for aspartase.

Synthesis and nucleic acid binding evaluation of a thyminyl l-diaminobutanoic acid-based nucleopeptide.

Herein we present the synthesis of a l-diaminobutanoic acid (DABA)-based nucleopeptide (3), with an oligocationic backbone, realized by solid phase peptide synthesis using thymine-bearing DABA moieties alternating in the sequence with free ones. CD studies evidenced the ability of this oligothymine nucleopeptide, well soluble in aqueous solution, to alter the secondary structure particularly of complementary RNA (poly rA vs poly rU) and inosine-rich RNAs, like poly rI and poly rIC, and showed its preference in binding double vs single-stranded DNAs. Furthermore, ESI mass spectrometry revealed that 3 bound also G-quadruplex (G4) DNAs, with either parallel or antiparallel topologies (adopted in our experimental conditions by c-myc and tel22, respectively). However, it caused detectable changes only in the CD of c-myc (whose parallel G4 structure was also thermally stabilized by ~3 °C), while leaving unaltered the antiparallel structure of tel22. Interestingly, CD and UV analyses suggested that 3 induced a hybrid mixed parallel/antiparallel G4 DNA structure in a random-coil tel22 DNA obtained under salt-free buffer conditions. Titration of the random-coil telomeric DNA with 3 gave quantitative information on the stoichiometry of the obtained complex. Overall, the findings of this work suggest that DABA-based nucleopeptides are synthetic nucleic acid analogues potentially useful in antigene and antisense strategies. Nevertheless, the hexathymine DABA-nucleopeptide shows an interesting behaviour as molecular tool per se thanks to its efficacy in provoking G4 induction in random coil G-rich DNA, as well as for the possibility to bind and stabilize c-myc oncogene in a G4 structure.

[Production of L-2-aminobutyric acid from L-threonine using a trienzyme cascade].

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.

Covalent immobilization of recombinant Citrobacter koseri transaminase onto epoxy resins for consecutive asymmetric synthesis of L-phosphinothricin.

Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The Km value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

Integrating biocatalysis with chemocatalysis for selective transformations.

The integration of biocatalysis with chemocatalysis combines the excellent selectivity of the former with the robust reactivity of the latter and offers many advantages, such as lower cost, higher yield, enhanced selectivity, as well as less waste generation. In spite of the challenge of incompatibilities between different classes of catalysts, recent advances in synthetic chemistry and biology provide ample opportunities for multistep cascade transformations that combine biocatalysis and chemocatalysis. Herein, we review recent progress in merging biocatalysis with chemocatalysis, highlighting selected examples of photo-/electricity-driven biotransformations and recently developed strategies for addressing the catalyst incompatibility issue.

Radiosynthesis of the 11 C-methyl derivative of LBQ657 for PET investigation of the neprilysin inhibitor sacubitril.

Neprilysin, also known as neutral endopeptidase, is a cell surface membrane metalo-endopeptidase that cleaves various peptides. Altered neprilysin expression has been correlated with various cancers and cardiovascular diseases. In this work, we present the radiosynthesis of the novel O-11 C-methylated derivative of LBQ657 (a potent neprilysin inhibitor). (2R,4S)-5-(Biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid [11 C]methyl ester ([11 C]MeOLBQ) is an analog of sacubitril where the alkyl ester is a 11 C-methyl instead of an ethyl. [11 C]MeOLBQ was produced in a one-pot two-step synthesis. The O-11 C-methylation of the pentanoic acid part was done with [11 C]methyl triflate followed by the deprotection of the tert-butyl ester precursor in acidic conditions. [11 C]MeOLBQ ([11 C]7) was produced in 9.5 ± 2.5% RCY (25 ± 6% decay-corrected from [11 C]CO2 , n = 3) high molar activity 348 ± 100 GBq/µmol (9425 ± 2720 mCi/µmol) at EOS, in high chemical (>95%) and radiochemical (>99%) purities. The total synthesis time including HPLC purification and reformulation was 29 minutes. To our knowledge, this is the first PET-labeled analog of the clinically used NEP inhibitor sacubitril.

Synthesis of ß-Substituted γ-Aminobutyric Acid Derivatives through Enantioselective Photoredox Catalysis.

ß-Substituted chiral γ-aminobutyric acids feature important biological activities and are valuable intermediates for the synthesis of pharmaceuticals. Herein, an efficient catalytic enantioselective approach for the synthesis of ß-substituted γ-aminobutyric acid derivatives through visible-light-induced photocatalyst-free asymmetric radical conjugate additions is reported. Various ß-substituted γ-aminobutyric acid analogues, including previously inaccessible derivatives containing fluorinated quaternary stereocenters, were obtained in good yields (42-89 %) and with excellent enantioselectivity (90-97 % ee). Synthetically valuable applications were demonstrated by providing straightforward synthetic access to the pharmaceuticals or related bioactive compounds (S)-pregabalin, (R)-baclofen, (R)-rolipram, and (S)-nebracetam.

Efficient reductive amination process for enantioselective synthesis of L-phosphinothricin applying engineered glutamate dehydrogenase.

The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida (PpGluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of PpGluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, PpGluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that PpGluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop.

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (RP/SP). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (kon=174M-1s-1) was ca. 8-fold more potent than the d-isomer (kon=21.5M-1s-1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be SP. The SP-isomers inhibited human GGT (kon=21.5-174M-1s-1), while the RP-isomers were inactive even at concentrations of 0.1mM.

Discovery of Novel and Highly Selective Inhibitors of Calpain for the Treatment of Alzheimer's Disease: 2-(3-Phenyl-1H-pyrazol-1-yl)-nicotinamides.

Calpain overactivation has been implicated in a variety of pathological disorders including ischemia/reperfusion injury, cataract formation, and neurodegenerative diseases such as Alzheimer's disease (AD). Herein we describe our efforts leading to the identification of ketoamide-based 2-(3-phenyl-1H-pyrazol-1-yl)nicotinamides as potent and reversible inhibitors of calpain with high selectivity versus related cysteine protease cathepsins, other proteases, and receptors. Broad efficacy in a set of preclinical models relevant to AD suggests that inhibition of calpain represents an attractive approach with potential benefit for the treatment of AD.

Discovery and Structure-Activity Relationships of the Neoseptins: A New Class of Toll-like Receptor-4 (TLR4) Agonists.

Herein, we report studies leading to the discovery of the neoseptins and a comprehensive examination of the structure-activity relationships (SARs) of this new class of small-molecule mouse Toll-like receptor 4 (mTLR4) agonists. The compounds in this class, which emerged from screening an α-helix mimetic library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are easy to produce and structurally manipulate, exhibit exquisite SARs, are nontoxic, and elicit improved and qualitatively different responses compared to lipopolysaccharide, even though they share the same receptor.

Synthesis of a Precursor to Sacubitril Using Enabling Technologies.

An efficient preparation of a precursor to the neprilysin inhibitor sacubitril is described. The convergent synthesis features a diastereoselective Reformatsky-type carbethoxyallylation and a rhodium-catalyzed stereoselective hydrogenation for installation of the two key stereocenters. Moreover, by integrating machine-assisted methods with batch processes, this procedure allows a safe and rapid production of the key intermediates which are promptly transformed to the target molecule (3·HCl) over 7 steps in 54% overall yield.

Synthesis of nisin AB dicarba analogs using ring-closing metathesis: influence of sp(3) versus sp(2) hybridization of the α-carbon atom of residues dehydrobutyrine-2 and dehydroalanine-5 on the lipid II binding affinity.

Herein the synthesis of two nisin AB dicarba analogs is described, focusing on amino acid modifications at positions 2 and 5. The nisin mimics were synthesized by a combination of solid phase synthesis of the linear peptides, followed by macrocyclization via ring-closing metathesis and fragment assembly by means of solution phase chemistry. The two N-terminal nisin AB-fragment mimics contain either the native dehydrobutyrine (Dhb)/dehydroalanine (Dha) amino acid residues or alanine at position 2 and 5, respectively. The native dehydrobutyrine at position 2 and dehydroalanine at position 5 were introduced as their precursors, namely threonine and serine, respectively, and subsequent dehydration was carried out by EDCI/CuCl as the condensing agent. Both AB-fragment mimics were analyzed in a lipid II binding assay and it was found that the Ala2/Ala5 AB-mimic (2) showed a reduced activity, while the Dhb2/Dha5 AB-mimic (3) was as active as the native AB-fragment (1).

Design, synthesis, and pharmacological evaluation of highly potent and selective dipeptidyl peptidase-4 inhibitors.

The optimization of a series of fused ß-homophenylalanine inhibitors of dipeptidyl peptidase-4 (DPP-4) is described. Modification on the P2-binding moiety of 6 (IC50 = 10 nM) led to the discovery of ß-homophenylalanine derivatives containing pyrrolidin-2-ylmethyl amides. The introduction of a sulfamine in the meta position of the phenyl ring improved the potency against DPP-4 (6-12-fold increase). Compound 14k showed DPP-4 inhibitory activity with an IC50 value of 0.87 nM. Meanwhile, in vivo experiments exhibited that 14h had an efficiency comparable to sitagliptin at the dose of 10 mg/kg.

Preparation of conformationally restricted ß(2,2)- and ß(2,2,3)-amino esters and derivatives containing an all-carbon quaternary center.

ß-Amino acids are routinely incorporated into peptidic drugs to increase their stability and to incur conformational biases. However, the synthesis of highly substituted ß-amino acids still represents a great challenge. A new approach to their preparation is reported involving a Vilsmeier-Haack reaction with nonaromatic carbon nucleophiles. The highly challenging preparation of contiguous tertiary and all-carbon quaternary centers was successfully used to generate several ß(2,2,3)-amino esters, such as derivatives of homoproline, homoalanine, and homopipecolinic esters.

Coimmobilization of l-methioninase and glutamate dehydrogenase: Novel approach for L-homoalanine synthesis.

L-Homoalanine, a nonnatural amino acid that is rarely found in human and microorganisms, is used in the synthesis of various medically pivotal antiepileptic drugs and antituberculosis compounds. l-Homoalanine can be synthesized by different enzymatic approaches. In this article, the synthesis of l-homoalanine from l-methionine was explored by coimmobilization of Aspergillus flavipes l-methioninase (AfMETase) and glutamate dehydrogenase (GDH) on polyacrylamide and chitosan. Polyacrylamide coimmobilized AfMETase and GDH displayed a maximum reactivity for the synthesis of homoalanine from l-methionine. The chitosan-coimmobilized AfMETase and GDH retain about 70% of their initial activity of l-homoalanine production by the fifth catalytic reusability cycle as compared with 50% for polyacrylamide coimmobilizate. Catalytic conditions were optimized for the maximum yield of homoalanine. Homoalanine was purified by cationic and anionic chromatographs and the proton nuclear magnetic resonance (H-NMR) analysis of the lyophilized sample displayed a unique chemical structure identical to the authentic homoalanine. Using dependable dual action of AfMETase and GDH immobilized on a solid support is a novel approach for in vitro enzymatic synthesis of l-homoalanine from l-methionine, and the immobilized enzymes can be reused many times without any significant loss of their activities.

Synthesis and evaluation of antineurotoxicity properties of an amyloid-ß peptide targeting ligand containing a triamino acid.

Peptide-like compounds containing an arginine have been shown to bind and stabilize the central helix of the Alzheimer's disease related amyloid-ß peptide (Aß) in an α-helical conformation, thereby delaying its aggregation into cytotoxic species. Here we study a novel Aß targeting ligand AEDabDab containing the triamino acid, N(γ)-(2-aminoethyl)-2,4-diaminobutanoic (AEDab) acid. The new AEDab triamino acid carries an extra positive charge in the side chain and is designed to be incorporated into a ligand AEDabDab where the AEDab replaces an arginine moiety in a previously developed ligand Pep1b. This is done in order to increase the Aß-ligand interaction, and molecular dynamics (MD) simulation of the stability of the Aß central helix in the presence of the AEDabDab ligand shows further stabilization of the helical conformation of Aß compared to the previously reported Pep1b as well as compared to the AEOrnDab ligand containing an N(δ)-(2-aminoethyl)-2,5-diaminopentanoic acid unit which has an additional methylene group. To evaluate the effect of the AEDabDab ligand on the Aß neurotoxicity the AEDab triamino acid building block is synthesized by reductive alkylation of N-protected-glycinal with α-amino-protected diaminobutanoic acid, and the Aß targeting ligand AEDabDab is prepared by solid-phase synthesis starting with attachment of glutarate to the Wang support. Replacement of the arginine residue by the AEDab triamino acid resulted in an improved capability of the ligand to prevent the Aß1-42 induced reduction of gamma (γ) oscillations in hippocampal slice preparation.