Stanton F. McHardy

A Novel Small Molecule Inhibitor of Candida albicans Biofilm Formation, Filamentation and Virulence with Low Potential for the Development of Resistance

Background/Objectives:Candida albicans is the principal causative agent of candidiasis, the most common fungal infection in humans. Candidiasis represents the third-to-fourth most frequent nosocomial infection worldwide, as this normal commensal of humans causes opportunistic infections in an expanding population of immune- and medically compromised patients. These infections are frequently associated with biofilm formation, which complicates treatment and contributes to unacceptably high mortality rates.Methods:To address the pressing need for new antifungals, we have performed a high-content screen of 20,000 small molecules in a chemical library (NOVACore) to identify compounds that inhibit C. albicans biofilm formation, and conducted a series of follow-up studies to examine the in vitro and in vivo activity of the identified compounds.Results:The screen identified a novel series of diazaspiro-decane structural analogs that were largely represented among the bioactive compounds. Characterization of the leading compound from this series indicated that it inhibits processes associated with C. albicans virulence, most notably biofilm formation and filamentation, without having an effect on overall growth or eliciting resistance. This compound demonstrated in vivo activity in clinically relevant murine models of both invasive and oral candidiasis and as such represents a promising lead for antifungal drug development. Furthermore, these results provide proof of concept for the implementation of antivirulence approaches against C. albicans and other fungal infections that would be less likely to foster the emergence of resistance.

Targeting Candida albicans filamentation for antifungal drug development

ABSTRACT Candida albicans remains the main etiological agent of candidiasis, as this otherwise normal commensal of humans is capable of causing active infection in immune- and medically-compromised patients. The high morbidity and mortality rates associated with candidiasis, coupled with the emergence of drug resistance demand the development of novel therapeutic strategies. However, there is a paucity of selective targets that can be exploited in the development of new antifungals. Contrary to conventional antibiotics that kill or curtail growth, specifically targeting virulence mechanisms represents an attractive option for antifungal drug development. In C. albicans, a growing body of research over the last few decades has provided important insights into its virulence factors and their contribution to the pathogenesis of candidiasis. Of these, filamentation is the one that has received the most attention and perhaps shows the most promise as a target for new anti-virulence strategies to combat C. albicans infections.

Design and discovery of a selective small molecule κ opioid antagonist (2-methyl- n -((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl) methyl)propan-1-amine, PF-4455242)

By use of parallel chemistry coupled with physicochemical property design, a series of selective κ opioid antagonists have been discovered. The parallel chemistry strategy utilized key monomer building blocks to rapidly expand the desired SAR space. The potency and selectivity of the in vitro κ antagonism were confirmed in the tail-flick analgesia model. This model was used to build an exposure-response relationship between the κ K(i) and the free brain drug levels. This strategy identified 2-methyl-N-((2-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242, which entered phase 1 clinical testing and has demonstrated target engagement in healthy volunteers.

Recent advances in acetylcholinesterase Inhibitors and Reactivators: an update on the patent literature (2012-2015)

ABSTRACT Introduction: Acetylcholinesterase (AChE) is the major enzyme that hydrolyzes acetylcholine, a key neurotransmitter for synaptic transmission, into acetic acid and choline. Mild inhibition of AChE has been shown to have therapeutic relevance in Alzheimer’s disease (AD), myasthenia gravis, and glaucoma among others. In contrast, strong inhibition of AChE can lead to cholinergic poisoning. To combat this, AChE reactivators have to be developed to remove the offending AChE inhibitor, restoring acetylcholine levels to normal. Areas covered: This article covers recent advances in the development of acetylcholinesterase modulators, including both inhibitors of acetylcholinesterase for the efforts in development of new chemical entities for treatment of AD, as well as re-activators for resurrection of organophosphate bound acetylcholinesterase. Expert opinion: Over the past three years, research efforts have continued to identify novel small molecules as AChE inhibitors for both CNS and peripheral diseases. The more recent patent activity has focused on three AChE ligand design areas: derivatives of known AChE ligands, natural product based scaffolds and multifunctional ligands, all of which have produced some unique chemical matter with AChE inhibition activities in the mid picomolar to low micromolar ranges. New AChE inhibitors with polypharmacology or dual inhibitory activity have also emerged as highlighted by new AChE inhibitors with dual activity at L-type calcium channels, GSK-3, BACE1 and H3, although most only show low micromolar activity, thus further research is warranted. New small molecule reactivators of organophosphate-inhibited AChE have also been disclosed, which focused on the design of neutral ligands with improved pharmaceutical properties and blood-brain barrier (BBB) penetration. Gratifyingly, some research in this area is moving away from the traditional quaternary pyridinium oximes AChE reactivators, while still employing the necessary reactivation group (oximes). However, selectivity over inhibition of native AChE enzyme, effectiveness of reactivation, broad-spectrum reactivation against multiple organophosphates and reactivation of aged-enzyme continue to be hurdles for this area of research.

Structure–Activity Relationship for Small Molecule Inhibitors of Nicotinamide N-Methyltransferase

Nicotinamide N-methyltransferase (NNMT) is a fundamental cytosolic biotransforming enzyme that catalyzes the N-methylation of endogenous and exogenous xenobiotics. We have identified small molecule inhibitors of NNMT with >1000-fold range of activity and developed comprehensive structure-activity relationships (SARs) for NNMT inhibitors. Screening of N-methylated quinolinium, isoquinolinium, pyrididium, and benzimidazolium/benzothiazolium analogues resulted in the identification of quinoliniums as a promising scaffold with very low micromolar (IC50 ∼ 1 μM) NNMT inhibition. Computer-based docking of inhibitors to the NNMT substrate (nicotinamide)-binding site produced a robust correlation between ligand-enzyme interaction docking scores and experimentally calculated IC50 values. Predicted binding orientation of the quinolinium analogues revealed selective binding to the NNMT substrate-binding site residues and essential chemical features driving protein-ligand intermolecular interactions and NNMT inhibition. The development of this new series of small molecule NNMT inhibitors direct the future design of lead drug-like inhibitors to treat several metabolic and chronic disease conditions characterized by abnormal NNMT activity.

Design, synthesis, and characterization of novel, nonquaternary reactivators of GF-inhibited human acetylcholinesterase.

The goal of this research was to identify structurally novel, non-quaternarypyridinium reactivators of GF (cyclosarin)-inhibited hAChE that possess the capacity to mediate in vitro reactivation of GF-inhibited human acetylcholinesterase (hAChE). New compounds were designed, synthesized and assessed in GF-inhibited hAChE assays. Structure activity relationships for AChE binding and reactivation of GF-inhibited hAChE were developed. Lead compounds from two different chemical series, represented by compounds 17 and 38, displayed proficient in vitro reactivation of GF-inhibited hAChE, while also possessing low inhibition of native enzyme.

18F-FDG microPET imaging detects early transient response to an IGF1R inhibitor in genetically engineered rhabdomyosarcoma models

Alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) are among the most common and most treatment resistant soft tissue sarcomas of childhood. Here, we evaluated the potential of 18F‐Fluorodeoxyglucose (FDG) as a marker of therapeutic response to picropodophyllin (PPP), an IGF1R inhibitor, in a conditional mouse model of ARMS and a conditional model of ERMS/undifferentiated pleomorphic sarcoma (UPS).

Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinsons disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.

Tyrosine phosphorylation regulates ERβ ubiquitination, protein turnover, and inhibition of breast cancer

Unlike estrogen receptor α (ERα) that predominantly promotes hormone-dependent breast tumor growth, ERβ exhibits antitumor effects in a variety of cancer types. We recently identified a phosphotyrosine residue in ERβ, but not ERα, that dictates ERβ transcriptional activity and antitumor function. We show here that this ER isotype-specific phosphotyrosine switch is important for regulating ERβ activity in cell proliferation, migration, and invasion. At the mechanistic level, phosphorylated ERβ, which recruits transcriptional coactivator p300, is in turn targeted by p300 for ubiquitination and proteasome-dependent protein turnover. Furthermore, ERβ-specific agonists such as S-equol enhance ERβ phosphorylation, suggesting a crosstalk between ligand- and posttranslational modification-dependent ERβ activation. Inhibition of xenograft tumor growth by S-equol is associated with reduced tumor Ki-67 expression and elevated ERβ tyrosine phosphorylation. Taken together, our data support the notion that phosphotyrosine-dependent ERβ signaling is an attractive target for anticancer treatment.

Molecular dynamics study of human carbonic anhydrase II in complex with Zn2+ and acetazolamide on the basis of all-atom force field simulations

Human carbonic anhydrase II (hCAII) represents an ultimate example of the perfectly efficient metalloenzymes, which is capable of catalyzing the hydration of carbon dioxide with a rate approaching the diffusion controlled limit. Extensive experimental studies of this physiologically important metalloprotein have been done to elucidate the fundamentals of its enzymatic actions: what residues anchor the Zn(2+) (or another divalent cation) at the bottom of the binding pocket; how the relevant residues work concertedly with the divalent cation in the reversible conversions between CO2 and HCO3(-); what are the protonation states of the relevant residues and acetazolamide, an inhibitor complexed with hCAII, etc. In this article, we present a detailed computational study on the basis of the all-atom CHARMM force field where Zn(2+) is represented with a simple model of divalent cation using the transferrable parameters available from the current literature. We compute the hydration free energy of Zn(2+), the characteristics of hCAII-Zn(2+) complexation, and the absolute free energy of binding acetazolamide to the hCAII-Zn(2+) complex. In each of these three problems, our computed results agree with the experimental data within the known margin of error without making any case-by-case adjustments to the parameters. The quantitatively accurate insights we gain in this all-atom molecular dynamics study should be helpful in the search and design of more specific inhibitors of this and other carbonic anhydrases.