In silico and pharmacological screenings identify novel serine racemase inhibitors

d-Serine is a coagonist of the N-methyl-d-aspartate (NMDA)-type glutamate receptor and its biosynthesis is catalyzed by serine racemase (SR). The overactivation of the NMDA receptor has been implicated in the development of neurodegenerative diseases, strokes, and epileptic seizures, thus, the inhibitors of SR have potential against these pathological states. Here, we have developed novel inhibitors of SR by in silico screening and in vitro enzyme assay. The newly developed inhibitors have lower IC₅₀ value comparing with that of malonate, one of the standard SR inhibitor. The structural features of novel inhibitors suggest the importance of central amide structure having a phenoxy substituent in their structure for the SR inhibitory activity. The present findings suggest the importance and rational development of new drugs for diseases of NMDAR overactivation.     

Development and validation of high-throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors

Poly(ADP-ribose) polymerase-1 and -2 (PARP1/2) are two key facilitators of DNA repair and are implicated in the pathogenesis of cancers and several chronic diseases. Inhibitors of PARP1/2 have shown powerful therapeutic effects in the treatment of cancer, cerebral ischemia, and inflammation. In addition, evidence from several studies suggests unique functions for PARP2 in genome surveillance, spermatogenesis, adipogenesis, and T cell development, and PARP2-specific inhibitors might have many other applications. To acquire PARP1/2 inhibitors, many high-throughput screening (HTS) assays for PARP1 inhibitors have been developed. However, detailed screening assays for PARP2 inhibitors have not been reported. Herein, three HTS assays for PARP2 inhibitors were developed and validated with reference inhibitors in each case. The results suggest that the HTS assays for PARP2 inhibitors using chemical quantification of NAD⁺, biotin-based quantification of PAR, and ELISA quantification of PAR are sensitive, robust, and cost effective.     

Multiple e-Pharmacophore modeling to identify a single molecule that could target both streptomycin and paromomycin binding sites for 30S ribosomal subunit inhibition

The bacterial ribosome is an established target for anti-bacterial therapy since decades. Several inhibitors have already been developed targeting both defined subunits (50S and 30S) of the ribosome. Aminoglycosides and tetracyclines are two classes of antibiotics that bind to the 30S ribosomal subunit. These inhibitors can target multiple active sites on ribosome that have a complex structure. To screen putative inhibitors against 30S subunit of the ribosome, the crystal structures in complex with various known inhibitors were analyzed using pharmacophore modeling approach. Multiple active sites were considered for building energy-based three-dimensional (3D) pharmacophore models. The generated models were validated using enrichment factor on decoy data-set. Virtual screening was performed using the developed 3D pharmacophore models and molecular interaction towards the 30S ribosomal unit was analyzed using the hits obtained for each pharmacophore model. The hits that were common to both streptomycin and paromomycin binding sites were identified. Further, to predict the activity of these hits a robust 2D-QSAR model with good predictive ability was developed using 16 streptomycin analogs. Hence, the developed models were able to identify novel inhibitors that are capable of binding to multiple active sites present on 30S ribosomal subunit.     

Disubstituted pyrimidines as Lck inhibitors

We have developed a family of 4-benzimidazolyl-N-piperazinethyl-pyrimidin-2-amines that are subnanomolar inhibitors of Lck. A subset of these Lck inhibitors, with heterocyclic substituents at the benzimidazole C5, are also low-nanomolar inhibitors of cellular IL2 release.     

Discovery of aminoheterocycles as a novel β-secretase inhibitor class: pH dependence on binding activity part 1

We have developed a novel series of heteroaromatic BACE-1 inhibitors. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, we have discovered a pH dependent effect on the binding affinity as a result of the intrinsic pK_a of these inhibitors and the pH of the BACE-1 enzyme binding assay.     

Mechanism and inhibition of BRAF kinase

The role of BRAF in tumor initiation has been established, however, the precise mechanism of autoinhibition has only been illustrated recently by several structural studies. These structures uncovered the basis by which the regulatory domains engage in regulating the activity of BRAF kinase domain, which lead to a more complete picture of the regulation cycle of RAF kinases. Small molecule BRAF inhibitors developed specifically to target BRAF^V600E have proven effective at inhibiting the most dominant BRAF mutant in melanomas, but are less potent against other BRAF mutants in RAS-driven diseases due to paradoxical activation of the MAPK pathway. A variety of new generation inhibitors that do not show paradoxical activation have been developed. Alternatively, efforts have begun to develop inhibitors targeting the dimer interface of BRAF. A deeper understanding of BRAF regulation together with more diverse BRAF inhibitors will be beneficial for drug development in RAF or RASdriven cancers.     

Prostaglandin endoperoxide H synthase inhibitors and other tocolytics in preterm labour

Preterm delivery (<37 weeks of gestation) is the major obstetrical complication in developed countries, yet attempts to delay labour and prolong pregnancy have largely been unsuccessful. One of the many reasons it is so difficult to prevent preterm birth is that the nature of preterm labour changes as a function of gestational age, maternal lifestyle factors or infection, to list a few of the reasons. The inhibitors of prostaglandin endoperoxide H synthase (PGHS), known as the Non-steroidal Antiinflammatory Drugs, have been viewed with interest as tocolytics with promising effectiveness under most conditions of preterm labour. Three isoforms of PGHS exist; the first two, PGHS-1 and -2, have been studied for their catalytic activity, X-ray crystallographic structure, and physiological roles in the adult and the foetus. Mixed inhibitors and isoform-specific inhibitors of PGHS have been developed, and their roles in delaying preterm labour are examined and compared to other tocolytics.     

Implementation of high-content assay for inhibitors of mitogen-activated protein kinase phosphatases

Small molecule inhibitors of protein tyrosine kinases have become both powerful chemical probes of biological processes and clinically effective therapeutics. In contrast, few small molecule inhibitors of protein tyrosine phosphatases have been identified and none are currently approved for clinical use. New cell-based high-content methods have been developed that should enable investigators to probe for selective inhibitors of diseases-relevant protein phosphatases. Details of these methods are described herein.     

Development of a Microemulsion Formulation for Antimicrobial SecA Inhibitors

In our previous study, we have identified five antimicrobial small molecules via structure based design, which inhibit SecA of Candidatus Liberibacter asiaticus (Las). SecA is a critical protein translocase ATPase subunit and is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In this study, eleven compounds were identified using similarity search method based on the five lead SecA inhibitors identified previously. The identified SecA inhibitors have poor aqueous solubility. Thus a microemulsion master mix (MMX) was developed to address the solubility issue and for application of the antimicrobials. MMX consists of N-methyl-2-pyrrolidone and dimethyl sulfoxide as solvent and co-solvent, as well as polyoxyethylated castor oil, polyalkylene glycol, and polyoxyethylene tridecyl ether phosphate as surfactants. MMX has significantly improved the solubility of SecA inhibitors and has no or little phytotoxic effects at concentrations less than 5.0% (v/v). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the SecA inhibitors and streptomycin against eight bacteria including Agrobacterium tumefaciens, Liberibacter crescens, Rhizobium etli, Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti phylogenetically related to Las were determined using the broth microdilution method. MIC and MBC results showed that the 16 SecA inhibitors have antibacterial activities comparable to that of streptomycin. Overall, we have identified 11 potent SecA inhibitors using similarity search method. We have developed a microemulsion formulation for SecA inhibitors which improved the antimicrobial activities of SecA inhibitors.     

2-Trifluoroacetylthiophenes, a novel series of potent and selective class II histone deacetylase inhibitors

The identification of class II HDAC inhibitors has been hampered by lack of efficient enzyme assays, in the preceding paper two assays have been developed to improve the efficiency of these enzymes: mutating an active site histidine to tyrosine, or by the use of a trifluoroacetamide lysine substrate, allowing screening to identify class II HDAC inhibitors. Herein, 2-trifluoroacetylthiophenes have been demonstrated to inhibit class II HDACs, resulting in the development of a series of 5-(trifluoroacetyl)thiophene-2-carboxamides as novel, potent and selective class II HDAC inhibitors. X-ray crystal structures of the HDAC 4 catalytic domain with a bound inhibitor demonstrate these compounds are active site inhibitors and bind in their hydrated form.     

SH2 domain inhibition: a problem solved?

The past two years have witnessed a number of significant advances in the design of SH2 inhibitors of both Src and Grb2. For Src, several non-peptide templates have been developed with high affinity, and one case, in the context of bone-binding phosphotyrosine bioisostere, has yielded an in vivo active antiresorptive agent. Similarly, high-affinity Grb2 SH2 inhibitors with novel phosphotyrosine replacements have now been reported that demonstrate, for the first time, cellular activities consistent with an anticancer agent.     

Mechanism-based inhibitors of dopamine beta-hydroxylase

The copper-containing monooxygenase dopamine beta-hydroxylase catalyzes the hydroxylation of dopamine at the benzylic position to form norepinephrine. Mechanism-based inhibitors for dopamine beta-hydroxylase have been used as probes of the mechanism of catalysis. The variety of such inhibitors that have been developed for this enzyme can be divided into three groups: (i) those in which the inactivating species is formed by abstraction of a hydrogen atom to form a radical intermediate; (ii) those in which the inactivating species is formed by abstraction of an electron to form an epoxide-like intermediate; and (iii) those in which the product is the inactivating species. A mechanism consistent with inactivation by all three groups of inhibitors which proposes that hydroxylation of dopamine by dopamine beta-hydroxylase involves formation of a benzylic radical has been developed. The benzylic radical is formed by abstraction of a hydrogen atom from the substrate by a high-potential copper-oxygen species.     

Design, synthesis, and evaluation of potent and selective benzoyleneurea-based inhibitors of protein geranylgeranyltransferase-I

A series of novel protein geranylgeranyltransferase-I (PGGTase-I) inhibitors based on a benzoyleneurea scaffold has been synthesized. Using a benzoyleneurea scaffold as a mimetic for the central dipeptide (AA), we have developed CAAX peptidomimetic inhibitors that selectively block the activity of PGGTase-I over the closely related enzyme protein farnesyltransferase. In this new class of PGGTase-I inhibitors, compound (6c) with X=L-phenylalanine, displayed the highest inhibition activity against PGGTase-I with an IC50 value of 170 nM. The inhibitors described in this study represent novel and promising leads for the development of potent and selective inhibitors of mammalian PGGTase-I for potential application as antitumor agents.     

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.     

Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors

We have developed a fluorescently labeled probe for high-throughput screening of kinase inhibitors using fluorescence correlation spectroscopy. With this probe, we have successfully evaluated the inhibitory activities of known inhibitors of a model kinase, ASK1. Because the probe contains a general kinase inhibitor, staurosporine, we believe that this homogeneous, high-throughput, and simple method can be applied to the inhibitor screening of other kinases as well.     

RANTES衍生物与HIV-1进入抑制剂

HIV-1-1进入抑制剂的研究是近年来艾滋病药物研发领域的新热点,其中最受关注的是以CCR5为靶点的新药研发。CCR5是病毒进入细胞的主要辅助受体,在HIV-1进入宿主细胞过程中起着非常重要的作用。作为CCR5的天然配体,CC类的趋化因子RANTES、MIP-1α和MIP-1β都是极具潜力的HIV-1抑制剂,特别是有关对RANTES的定向设计的研究尤为引人关注,其目的是设计出一种既有很强的抗病毒能力而又不引发炎症反应的HIV-1拮抗剂。就RANTES衍生物应用于抑制HIV进入细胞方面的研究进行了综述。     

Drug discovery considerations in the development of covalent inhibitors

In recent years, the number of drug candidates with a covalent mechanism of action progressing through clinical trials or being approved by the FDA has increased significantly. And as interest in covalent inhibitors has increased, the technical challenges for characterizing and optimizing these inhibitors have become evident. A number of new tools have been developed to aid this process, but these have not gained wide-spread use. This review will highlight a number of methods and tools useful for prosecuting covalent inhibitor drug discovery programs.     

HDAC inhibitors in cancer biology: emerging mechanisms and clinical applications

Reversible acetylation mediated by histone deacetylases (HDACs) influences a broad repertoire of physiological processes, many of which are aberrantly controlled in tumor cells. As HDAC inhibition prompts tumor cells to enter apoptosis, small-molecule HDAC inhibitors have been developed as a new class of mechanism-based anti-cancer agent, many of which have entered clinical trials. Although the clinical picture is evolving and the precise utility of HDAC inhibitors remains to be determined, it is noteworthy that certain tumor types undergo a favorable response, in particular hematological malignancies. Vorinostat and romidepsin have been approved for treating cutaneous T-cell lymphoma in patients with progressive, persistent or recurrent disease. Here, we discuss developments in our understanding of molecular events that underlie the anti-cancer effects of HDAC inhibitors and relate this information to the emerging clinical picture for the application of these inhibitors in the treatment of cancer.