Broad-Spectrum Antifungal Agents: Fluorinated Aryl- and Heteroaryl-Substituted Hydrazones.

Fluorinated aryl- and heteroaryl-substituted monohydrazones displayed excellent broad-spectrum activity against various fungal strains, including a panel of clinically relevant Candida auris strains relative to a control antifungal agent, voriconazole (VRC). These monohydrazones displayed less hemolysis of murine red blood cells than that of VRC at the same concentrations, possessed fungicidal activity in a time-kill study, and exhibited no mammalian cell cytotoxicity. In addition, these monohydrazones prevented the formation of biofilms that otherwise block antibiotic effectiveness and did not trigger the development of resistance when exposed to C. auris AR Bank # 0390 over 15 passages.

N,N'-diaryl-bishydrazones in a biphenyl platform: Broad spectrum antifungal agents.

N,N'-Diaryl-bishydrazones of [1,1'-biphenyl]-3,4'-dicarboxaldehyde, [1,1'-biphenyl]-4,4'-dicarboxaldehyde, and 4,4'-bisacetyl-1,1-biphenyl exhibited excellent antifungal activity against a broad spectrum of filamentous and non-filamentous fungi. These N,N'-diaryl-bishydrazones displayed no antibacterial activity in contrast to previously reported N,N'-diamidino-bishydrazones and N-amidino-N'-aryl-bishydrazones. The leading candidate, 4,4'-bis((E)-1-(2-(4-fluorophenyl)hydrazono)ethyl)-1,1'-biphenyl, displayed less hemolysis of murine red blood cells at concentrations at or below that of a control antifungal agent (voriconazole), was fungistatic in a time-kill study, and possessed no mammalian cytotoxicity and no toxicity with respect to hERG inhibition.

Bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones: New classes of antibacterial/antifungal agents.

The emergence of multidrug-resistant bacterial and fungal strains poses a threat to human health that requires the design and synthesis of new classes of antimicrobial agents. We evaluated bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones for their antibacterial and antifungal activities against panels of Gram-positive/Gram-negative bacteria as well as fungi. We investigated their potential to develop resistance against both bacteria and fungi by a multi-step resistance-selection method, explored their potential to induce the production of reactive oxygen species, and assessed their toxicity. In summary, we found that these compounds exhibited broad-spectrum antibacterial and antifungal activities against most of the tested strains with minimum inhibitory concentration (MIC) values ranging from <0.5 to >500µM against bacteria and 1.0 to >31.3µg/mL against fungi; and in most cases, they exhibited either superior or similar antimicrobial activity compared to those of the standard drugs used in the clinic. We also observed minimal emergence of drug resistance to these newly synthesized compounds by bacteria and fungi even after 15 passages, and we found weak to moderate inhibition of the human Ether-à-go-go-related gene (hERG) channel with acceptable IC50 values ranging from 1.12 to 3.29µM. Overall, these studies show that bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones are potentially promising scaffolds for the discovery of novel antibacterial and antifungal agents.

Synthesis of a norcantharidin-tethered guanosine: Protein phosphatase-1 inhibitors that change alternative splicing.

Phosphorylation and dephosphorylation of splicing factors play a key role in pre-mRNA splicing events, and cantharidin and norcantharidin analogs inhibit protein phosphatase-1 (PP1) and change alternative pre-mRNA splicing. Targeted inhibitors capable of selectively inhibiting PP-1 could promote exon 7 inclusion in the survival-of-motorneuron-2 gene (SMN2) and shift the proportion of SMN2 protein from a dysfunctional to a functional form. As a prelude to the development of norcantharidin-tethered oligonucleotide inhibitors, the synthesis a norcantharidin-tethered guanosine was developed in which a suitable tether prevented the undesired cyclization of norcantharidin monoamides to imides and possessed a secondary amine terminus suited to the synthesis of oligonucleotides analogs. Application of this methodology led to the synthesis of a diastereomeric mixture of norcantharidin-tethered guanosines, namely bisammonium (1R,2S,3R,4S)- and (1S,2R,3S,4R)-3-((4-(2-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl)oxy)oxidophosphoryl)oxy)ethyl)-phenethyl)(methyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylate, which showed activity in an assay for SMN2 pre-mRNA splicing.

Selenoglycoproteins attenuate adhesion of tumor cells to the brain microvascular endothelium via a process involving NF-κB activation.

Selenium-containing compounds and selenized yeast have anticancer properties. In order to address possible mechanisms involved in these effects, selenoglycoproteins (SGPs) were extracted from selenium-enriched yeast at pH 4.0 and 6.5 (the fractions are called SGP40 and SGP65, respectively), followed by evaluation of their impact on the interactions of lung and breast tumor cells with human brain microvascular endothelial cells (HBMECs). Extracted SGPs, especially SGP40, significantly inhibited adhesion of tumor cells to HBMECs and their transendothelial migration. Because the active components of SGPs are unknown, small selenium-containing compounds [leucyl-valyl-selenomethionyl-arginine (LVSe-MR) and methylselenoadenosine (M-Se-A)], which are normally present in selenized yeast, were introduced as additional treatment groups. Treatment of HBMECs with SGP40, LVSe-MR and M-Se-A induced changes in gene signatures, which suggested a central involvement of nuclear factor (NF)-κB-dependent pathway. These observations were confirmed in the subsequent analysis of NF-κB DNA binding activity, quantitative measurements of the expression of selected genes and proteins, and tumor cell adhesion assay with a specific NF-κB inhibitor as the additional treatment factor. These findings indicate that specific organic selenium-containing compounds have the ability to inhibit tumor cell adhesion to brain endothelial cells via down-regulation of NF-κB. SGPs appear to be more effective than small selenium-containing compounds, suggesting the role of not only selenium but also the glycoprotein component in the observed protective impact.

Tritium-labeled (E,E)-2,5-bis(4'-hydroxy-3'-carboxystyryl)benzene as a probe for ß-amyloid fibrils.

Accumulation of Aß in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aß production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aß40 and Aß42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[(3)H]-2,5-bis(4'-hydroxy-3'-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aß plaque and tau pathology in brain tissue and in vitro studies with synthetic Aß and tau fibrils.

Application of the 4D fingerprint method with a robust scoring function for scaffold-hopping and drug repurposing strategies.

Two factors contribute to the inefficiency associated with screening pharmaceutical library collections as a means of identifying new drugs: [1] the limited success of virtual screening (VS) methods in identifying new scaffolds; [2] the limited accuracy of computational methods in predicting off-target effects. We recently introduced a 3D shape-based similarity algorithm of the SABRE program, which encodes a consensus molecular shape pattern of a set of active ligands into a 4D fingerprint descriptor. Here, we report a mathematical model for shape similarity comparisons and ligand database filtering using this 4D fingerprint method and benchmarked the scoring function HWK (Hamza-Wei-Korotkov), using the 81 targets of the DEKOIS database. Subsequently, we applied our combined 4D fingerprint and HWK scoring function VS approach in scaffold-hopping and drug repurposing using the National Cancer Institute (NCI) and Food and Drug Administration (FDA) databases, and we identified new inhibitors with different scaffolds of MycP1 protease from the mycobacterial ESX-1 secretion system. Experimental evaluation of nine compounds from the NCI database and three from the FDA database displayed IC50 values ranging from 70 to 100 µM against MycP1 and possessed high structural diversity, which provides departure points for further structure-activity relationship (SAR) optimization. In addition, this study demonstrates that the combination of our 4D fingerprint algorithm and the HWK scoring function may provide a means for identifying repurposed drugs for the treatment of infectious diseases and may be used in the drug-target profile strategy.

Pentapeptide boronic acid inhibitors of Mycobacterium tuberculosis MycP1 protease.

Mycosin protease-1 (MycP1) cleaves ESX secretion-associated protein B (EspB) that is a virulence factor of Mycobacterium tuberculosis, and accommodates an octapeptide, AVKAASLG, as a short peptide substrate. Because peptidoboronic acids are known inhibitors of serine proteases, the synthesis and binding of a boronic acid analog of the pentapeptide cleavage product, AVKAA, was studied using MycP1 variants from Mycobacterium thermoresistible (MycP1mth), Mycobacterium smegmatis (MycP1msm) and M. tuberculosis (MycP1mtu). We synthesized the boropentapeptide, HAlaValLysAlaAlaB(OH)2 (1) and the analogous pinanediol PD-protected HAlaValLysAlaAlaBO2(PD) (2) using an Fmoc/Boc peptide strategy. The pinanediol boropentapeptide 2 displayed IC50 values 121.6±25.3 µM for MycP1mth, 93.2±37.3 µM for MycP1msm and 37.9±5.2 µM for MycP1mtu. Such relatively strong binding creates a chance for crystalizing the complex with 2 and finding the structure of the unknown MycP1 catalytic site that would potentially facilitate the development of new anti-tuberculosis drugs.

Novel mycosin protease MycP1 inhibitors identified by virtual screening and 4D fingerprints.

The rise of drug-resistant Mycobacterium tuberculosis lends urgency to the need for new drugs for the treatment of tuberculosis (TB). The identification of a serine protease, mycosin protease-1 (MycP1), as the crucial agent in hydrolyzing the virulence factor, ESX-secretion-associated protein B (EspB), potentially opens the door to new tuberculosis treatment options. Using the crystal structure of mycobacterial MycP1 in the apo form, we performed an iterative ligand- and structure-based virtual screening (VS) strategy to identify novel, nonpeptide, small-molecule inhibitors against MycP1 protease. Screening of ∼485,000 ligands from databases at the Genomics Research Institute (GRI) at the University of Cincinnati and the National Cancer Institute (NCI) using our VS approach, which integrated a pharmacophore model and consensus molecular shape patterns of active ligands (4D fingerprints), identified 81 putative inhibitors, and in vitro testing subsequently confirmed two of them as active inhibitors. Thereafter, the lead structures of each VS round were used to generate a new 4D fingerprint that enabled virtual rescreening of the chemical libraries. Finally, the iterative process identified a number of diverse scaffolds as lead compounds that were tested and found to have micromolar IC50 values against the MycP1 target. This study validated the efficiency of the SABRE 4D fingerprints as a means of identifying novel lead compounds in each screening round of the databases. Together, these results underscored the value of using a combination of in silico iterative ligand- and structure-based virtual screening of chemical libraries with experimental validation for the identification of promising structural scaffolds, such as the MycP1 inhibitors.

Natural and modified (1-->3)-beta-D-glucans in health promotion and disease alleviation.

A number of polysaccharides with beta-glycosidic linkage are widespread in nature in a variety of sources. All have a common structure and the (1-->3)-beta-D-glucan backbone is essential. They have attracted attention over the years because of their bioactive and medicinal properties. In many cases their functional role is a mystery, in others it is well established. Because of their insoluble chemical nature, particulate (1-->3)-beta-D-glucans are not suitable for many medical applications. Various methods of changing or modifying the beta-D-glucan chemical structure and transforming it to a soluble form have been published. The beta-D-glucan bioactive properties can be affected positively or negatively by such modifications. This review examines beta-glucan sources in nature, health effects and structure-activity relationships. It presents the current state of beta-D-glucan solubilization methods and discusses their effectiveness and application possibilities for the future.