Lipophilic Ligands Research Articles

Benzoxazolinone aryl sulfonamides as potent, selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model

Studies on human genetics have suggested that inhibitors of the Nav1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Nav1.7 inhibitors with excellent selectivity against the Nav1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Nav1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.

Discovery and Development of Allosteric Pantothenate Kinase Activators

The central position of coenzyme A (CoA) in intermediary metabolism and its role in human neurodegenerative disease, led us to develop chemical probes targeting pantothenate kinase (PANK), the regulatory enzyme that controls CoA biosynthesis. There are four mammalian PANK isoforms that exhibit tissue‐specific expression and differ in their sensitivity to feedback inhibition by CoA and its thioesters. A high‐throughput screen and further medicinal chemistry efforts identified a novel class of orally bioavailable PanK modulators called pantazines. Optimization of the series based on lipophilic ligand efficiency (LipE) culminated in PZ‐2891, a drug‐like molecule that binds to PANK3 with nM affinity. Enzyme kinetics, biophysical studies, and X‐ray crystallography demonstrate that PZ‐2891 binds to the pantothenate substrate site of the PANK3•ATP•Mg2+ complex and extends across the dimer interface to form an induced fit, high‐affinity quaternary complex. PANK3 is a highly cooperative dimer and both protomer active sites simultaneously switch between the closed, active conformation that binds ATP, and the open, inactive conformation that binds acetyl‐CoA. PZ‐2891 inactivates the protomer to which it is bound and locks the opposite protomer in an active conformation that is refractory to feedback regulation by acetyl‐CoA. This allosteric property means that PZ‐2891 is a pantothenate‐dependent activator of CoA biosynthesis in cultured cells, and substantially increases intracellular CoA levels as a function of the media pantothenate supplement. Administration of PZ‐2891 to animals by oral gavage recapitulated the pantothenate‐dependent modulation of CoA levels in liver. PZ‐2891 dose‐response experiments also demonstrated the elevation of brain CoA illustrating the ability of PZ‐2891 to cross the blood brain barrier. PZ‐2891 and its inactive analogs are ready to be deployed as a toolkit to manipulate CoA levels in cultured cells and animals to analyze the role of CoA concentration in the control of intermediary metabolism. These nanomolar PANK allosteric effectors are being further developed as preclinical leads to validate PANK as a target to treat neurologic and metabolic disorders.Support or Funding InformationNIH, ALSAC/St. Jude

Can We Make Small Molecules Lean? Optimization of a Highly Lipophilic TarO Inhibitor.

We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure-activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.

In vitro membrane binding and protein binding (IAM MB/PB technology) to estimate in vivo distribution: applications in early drug discovery

<p class="ADMETabstracttext">The drug discovery process can be accelerated by chromatographic profiling of the analogs to model in vivo distribution and the major non-specific binding. A balanced potency and chromatographically determined membrane and protein binding (IAM MB/PB) data enable selecting drug discovery compounds for further analysis that have the highest probability to show the desired in vivo distribution behavior for efficacy and reduced chance for toxicity. Although the basic principles of the technology have already appeared in numerous publications, the lack of standardized procedures limited its widespread applications especially in academia and small drug discovery biotech companies. In this paper, the standardized procedures are described that has been trademarked as Regis IAM MB/PB Technology®. Comparison between the Drug Efficiency Index (DEI=pIC50-logVdu+2) and generally used Ligand Lipophilicity Efficiency (LLE) has been made, demonstrating the advantage of measured IAM and HSA binding over calculated log P. The power of the proposed chromatographic technology is demonstrated using the data of marketed drugs.</p>

Targeting the dengue β-OG with serotype-specific alkaloid virtual leads

The dengue envelope β-OG pocket is a crucial hinge for mediating virus-host fusion via conformational changes in the envelope to the fusion-competent form. The β-OG pocket is a small molecule target site for inhibition of virus-host fusion. As of date, the only structure of the β-OG pocket known is of serotype 2. Studies of β-OG inhibition by small molecules primarily target viral serotype 2. Envelope and β-OG sequence alignments, reveal dissimilarities across serotypes. In light of protein sequence-structure-function correlation, sequence variations suggest serotypic variations in β-OG druggability. This, together with the fact that dengue viral proteins do have serotype-specific variations of structure and function, lead to the study of the serotype-specificity of the dengue β-OG ligand binding behaviour. β-OG druggability was compared using comparative models of envelope proteins containing the β-OG pocket in four serotypes of the dengue virus. β-OG ligand binding was found to vary with respect to hydrophobicity, hydrophilicity, hydrogen bonding, van der Waals interactions with ligands and tightness of the binding site. The study also reports serotype-specific virtual leads identified from a library of 9175 alkaloids, using a consensus docking and scoring approach. The docking algorithms of Glide SP and XP, together with the Lamarckian genetic algorithm were employed for consensus docking. For consensus scoring, the Glide empirical score was employed along with the scoring function of AutoDock. A multi-dimensional lead optimisation approach was performed for optimising affinity, ligand efficiency, lipophilic ligand efficiency, ADMET and molecular torsional strains. The study proposes the serotype-specific inhibition of the β-OG for an effective inhibition of virus-host fusion, in contrast to a pan inhibitor.

Lipocalin-Type Prostaglandin D Synthase Possesses Two Binding Sites for its Product

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) belongs to the lipocalin superfamily which consists of transporter proteins for lipophilic ligands in the extracellular space, and is referred to as the PGD2-synthesizing enzyme, which is responsible for sleep regulation. Recently, we found that L-PGDS binds, not only to its substrate, PGH2, but also to its product, PGD2. However, the mechanism responsible for the binding of L-PGDS to its product, PGD2, remains unclear. In the present study, to address this issue, we investigated the interactions between L-PGDS and its ligand, such as PGD2, PGF2α and PGE2.

Finely Tuned Asymmetric Platinum(IV) Anticancer Complexes: Structure-Activity Relationship and Application as Orally Available Prodrugs.

Platinum(IV) bis-carboxylates are highly versatile prodrug scaffolds with different axial ligands that can be functionalized while keeping the platinum(II) pharmacophore intact. Using a sequential acylation strategy, we developed a class of PtIV prodrugs of cisplatin with contrasting lipophilic and hydrophilic ligands. We investigated their stability, reduction rates, lipophilicity, aqueous solubility, and antiproliferative efficacies, and assessed for correlations among the parameters that could be useful in drug design. We showed that compounds with high lipophilicity result in better antiproliferative effects in vitro and in vivo, with one of the three compounds tested showing better efficacy than satraplatin against an animal model of colorectal cancer, owing to its higher solubility and lower reduction rates. Our asymmetric PtIV prodrugs may pave the way for a highly predictable, fine-tuned class of orally available PtIV prodrugs for the treatment of colorectal cancer.

Nuclear Receptors in Skeletal Homeostasis.

Nuclear receptors are a family of transcription factors that can be activated by lipophilic ligands. They are fundamental regulators of development, reproduction, and energy metabolism. In bone, nuclear receptors enable bone cells, including osteoblasts, osteoclasts, and osteocytes, to sense their dynamic microenvironment and maintain normal bone development and remodeling. Our views of the molecular mechanisms in this process have advanced greatly in the past decade. Drugs targeting nuclear receptors are widely used in the clinic for treating patients with bone disorders such as osteoporosis by modulating bone formation and resorption rates. Deficiency in the natural ligands of certain nuclear receptors can cause bone loss; for example, estrogen loss in postmenopausal women leads to osteoporosis and increases bone fracture risk. In contrast, excessive ligands of other nuclear receptors, such as glucocorticoids, can also be detrimental to bone health. Nonetheless, the ligand-induced osteoprotective effects of many other nuclear receptors, e.g., vitamin D receptor, are still in debate and require further characterizations. This review summarizes previous studies on the roles of nuclear receptors in bone homeostasis and incorporates the most recent findings. The advancement of our understanding in this field will help researchers improve the applications of agonists, antagonists, and selective modulators of nuclear receptors for therapeutic purposes; in particular, determining optimal pharmacological drug doses, preventing side effects, and designing new drugs that are more potent and specific.

PdII2L4-type coordination cages up to three nanometers in size.

The utilization of large ligands in coordination-based self-assembly represents an attractive strategy for the construction of supramolecular assemblies more than two nanometers in size. However, the implementation of this strategy is hampered by the fact that the preparation of such ligands often requires substantial synthetic effort. Herein, we describe a simple one-step protocol, which allows large bipyridyl ligands with a bent shape to be synthesized from easily accessible and/or commercially available starting materials. The ligands were used to construct PdII2L4-type coordination cages of unprecedented size. Furthermore, we provide evidence that these cages may be stabilized by close intramolecular packing of lipophilic ligand side chains. Packing effects of this kind are frequently encountered in protein assemblies, but they are seldom used as a design element in metallasupramolecular chemistry.

Pyrazolo[3,4-d]pyrimidines as sigma-1 receptor ligands for the treatment of pain. Part 2: Introduction of cyclic substituents in position 4.

The replacement of acylamino by cyclic substituents in the position 4 of the pyrazolo[3,4-d]pyrimidine scaffold, led to highly active sigma-1 receptor (σ1R) ligands. Phenyl or pyrazolyl groups were the best in terms of affinity for the σ1R and the 4-(1-methylpyrazol-5-yl) derivative, 12f, was the most selective. Compound 12f is also one of the best σ1R ligands ever described in terms of lipophilic ligand efficiency, which translates into a good physicochemical and ADMET profile. In addition, 12f was identified as an antagonist of the σ1R in view of its potent antinociceptive profile in several pain models in mice.

Characterization of stem cell-derived liver and intestinal organoids as a model system to study nuclear receptor biology

Nuclear receptors (NRs) are ligand-activated transcription factors regulating a large variety of processes involved in reproduction, development, and metabolism. NRs are ideal drug targets because they are activated by lipophilic ligands that easily pass cell membranes. Immortalized cell lines recapitulate NR biology poorly and generating primary cultures is laborious and requires a constant need for donor material. There is a clear need for development of novel preclinical model systems that better resemble human physiology. Uncertainty due to technical limitations early in drug development is often the cause of preclinical drugs not reaching the clinic. Here, we studied whether organoids, mini-organs derived from the respective mouse tissue's stem cells, can serve as a novel model system to study NR biology and targetability. We characterized mRNA expression profiles of the NR superfamily in mouse liver, ileum, and colon organoids. Tissue-specific expression patterns were largely maintained in the organoids, indicating their suitability for NR research. Metabolic NRs Fxrα, Lxrα, Lxrβ, Pparα, and Pparγ induced expression of and binding to endogenous target genes. Transcriptome analyses of wildtype colon organoids stimulated with Rosiglitazone showed that lipid metabolism was the highest significant changed function, greatly mimicking the PPARs and Rosiglitazone function in vivo. Finally, using organoids we identify Trpm6, Slc26a3, Ang1, and Rnase4, as novel Fxr target genes. Our results demonstrate that organoids represent a framework to study NR biology that can be further expanded to human organoids to improve preclinical testing of novel drugs that target this pharmacologically important class of ligand activated transcription factors.

Intracellular allosteric antagonism of the CCR9 receptor.

Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.

Cationic Ru(η6‐p‐cymene) Complexes of 3‐Hydroxy‐4‐pyr(id)ones – Lipophilic Triphenylphosphine as Co‐Ligand Is Key to Highly Stable and Cytotoxic Anticancer Agents

Ru(arene)[3‐hydroxy‐4(1H)‐pyr(id)one]Cl complexes are known to be converted intodinuclear Ru(arene) compounds with three hydroxido bridges in aqueous solution. In an attempt to obtain anticancer Ru(arene) complexes of 3‐hydroxy‐4(1H)‐pyr(id)one ligands that are stable in aqueous solution, triphenylphosphine (PPh3) and 1,3,5‐triaza‐7‐phoshatricyclo[3.3.1.1]decane (pta) were introduced as co‐ligands instead of the chlorido ligand. This led to a series of cationic complexes that were characterized by using standard methods and X‐ray diffraction analysis. The pta complexes were found to be highly stable in aqueous solution for up to 120 h. While they were unreactive in the presence of l‐histidine (His) and l‐methionine (Met), l‐cysteine (Cys) induced cleavage of the 3‐hydroxy‐4(1H)‐pyr(id)one ligands from the Ru center. In vitro cytotoxicity assays against human cancer cell lines revealed that the complexes with the more lipophilic phosphine ligand were cytotoxic in the low‐micromolar concentration range, while the analogous pta compounds did not significantly inhibit cell proliferation.

Structure-Activity Relationships of 2-Sufonylpyrimidines as Quorum-Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS-QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure-activity relationships of 2-sulfonylpyrimidines, which were previously identified as dual-target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.

Acid-switched Eu(III) coordination inside reverse aggregates: Insights into a synergistic liquid-liquid extraction system

Determining the structure of complex solutions bearing metal ions is challenging, but crucial for developing important technologies such as liquid-liquid extraction for metal refining and separation purposes. Herein, the structure of an organic Eu(III) solution consisting a binary mixture of lipophilic ligands di-2-ethylhexyl phosphoric acid (HDEHP) and tetraoctyl diglycolamide (TODGA) in dodecane is studied using synchrotron small angle X-ray scattering (SAXS) and X-ray absorption fine structure spectroscopy (EXAFS). This system is of technological importance in f-element separation for nuclear fuel cycle applications, where extraction is controlled by varying nitric acid concentration. Extraction is promoted at low and high concentration, but is retarded at intermediate concentration, leading to a U-shaped function; the structural origins of which we investigate. At the nanoscale, the solution is apparently comprised of reverse micelles with polar cores of approximately 1nm in size, and these remain virtually unchanged as acid concentration is varied. Inside the polar cores, the coordination environment of Eu(III) switches from a 9-coordinate [Eu(TODGA)3]3+ motif at high acid, to a 6-coordinate HDEHP-dominated complex resembling Eu(HDEHP·DEHP)3 at low acid. The results show that extraction is controlled within the coordination sphere, where it is promoted under conditions that favor coordination of either one of the two organic ligands, but is retarded under conditions that encourage mixed complexes. Our results link solution structure with ion transport properties in a technologically-important liquid-liquid ion extraction system.

Developmental and Cell Cycle Quiescence Is Mediated by the Nuclear Hormone Receptor Coregulator DIN-1S in the Caenorhabditis elegans Dauer Larva

When faced with suboptimal growth conditions, Caenorhabditis elegans larvae can enter a diapause-like stage called "dauer" that is specialized for dispersal and survival. The decision to form a dauer larva is controlled by three parallel signaling pathways, whereby a compromise of TGFβ, cyclic guanosine monophosphate, or insulin/IGF-like signaling (ILS) results in dauer formation. Signals from these pathways converge on DAF-12, a nuclear hormone receptor that triggers the changes required to initiate dauer formation. DAF-12 is related to the vitamin D, liver-X, and androstane receptors, and like these human receptors, it responds to lipophilic hormone ligands. When bound to its ligand, DAF-12 acquires transcriptional activity that directs reproductive development, while unliganded DAF-12 forms a dauer-specifying complex with its interacting protein DIN-1S to regulate the transcription of genes required for dauer development. We report here that din-1S is required in parallel to par-4/LKB1 signaling within the gonad to establish cell cycle quiescence during the onset of the dauer stage. We show that din-1S is important for postdauer reproduction when ILS is impaired and is necessary for long-term dauer survival in response to reduced ILS. Our work uncovers several previously uncharacterized functions of DIN-1S in executing and maintaining many of the cellular and physiological processes required for appropriate dauer arrest, while also shedding light on the coordination of nuclear hormone signaling, the LKB1/AMPK signaling cascade, and ILS/TGFβ in the control of cell cycle quiescence and tissue growth: a key feature that is often misregulated in a number of hormone-dependent cancers.

Clickable 4-Oxo-β-lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes.

Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC, or 'click' chemistry) approach based on the 4-oxo-β-lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure-activity relationships set the basis to develop fluorescent and biotinylated activity-based probes as tools for molecular functional analysis. Attaching the tags to the 4-oxo-β-lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC50 values in the nanomolar range (56-118 nm) displayed by the probes. The nitrobenzoxadiazole (NBD)-based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity-based probes with promising applications in target validation and identification, as well as diagnostic tools.

Potentiometric Anion Selectivity and Analytical Applications of Polymer Membrane Electrodes Based on Novel Mn(III)‐ and Mn(IV)‐Salophen Complexes

AbstractNew Mn(III)‐L and Mn(IV)‐L complexes were prepared from the highly lipophilic salophen ligand (L): phenol 2,2′‐[(4,5‐dimethyl‐1,2‐phenylene)bis[(E)‐nitrilomethylidyne]]bis[4,6‐bis(1,1‐dimethylethyl). The prepared complexes were fully characterized and used for the construction of thiocyanate membrane electrodes. Optimized membrane electrodes contained 33.0 mg PVC, 66.0 mg o‐nitrophenyloctylether, 50 or 5 (mole %) tetrakis(trifluoromethyl)phenyl borate and 1 mg Mn(III)‐L (sensor 2) or Mn‐(IV)‐L (sensor 12), respectively. Such electrodes exhibited linear responses toward thiocynate in a concentration range of 10−1–10−5 M and detection limits of 8.3×10−6, 8.9×10−6 M for sensor 2 and 12, respectively. Optimized membrane electrodes exhbited high selectivty toward thiocayante compared to more lipophilic anions. The observed thiocyanate selectivity of the optimized membranes was confirmed by formation constant calculations for Mn(III)‐L and Mn(IV)‐L with SCN−, β=1014.1 and 1012.5, which was measured potentiometrically using the sandwich membrane method. Furthermore, computational study using DFT calculations was performed to at DFT/B3LYP level of theory to confirm the observed selectivity data. The response times were 3 and 0.5 min for low and high concentrations. The lifetimes of the optimized electrodes were ∼4–6 weeks. The analytical utility of the optimized membrane electrodes was demonstrated by the analysis of thiocyanate level in different saliva samples.

Probing Lipophilic Adamantyl Group as the P1-Ligand for HIV-1 Protease Inhibitors: Design, Synthesis, Protein X-ray Structural Studies, and Biological Evaluation.

A series of potent HIV-1 protease inhibitors with a lipophilic adamantyl P1 ligand have been designed, synthesized, and evaluated. We have developed an enantioselective synthesis of adamantane-derived hydroxyethylamine isosteres utilizing Sharpless asymmetric epoxidation as the key step. Various inhibitors incorporating P1-adamantylmethyl in combination with P2 ligands such as 3-(R)-THF, 3-(S)-THF, bis-THF, and THF-THP were examined. The S1' pocket was also probed with phenyl and phenylmethyl ligands. Inhibitor 15d, with an isobutyl P1' ligand and a bis-THF P2 ligand, proved to be the most potent of the series. The cLogP value of inhibitor 15d is improved compared to inhibitor 2 with a phenylmethyl P1-ligand. X-ray structural studies of 15d, 15h, and 15i with HIV-1 protease complexes revealed molecular insight into the inhibitor-protein interaction.

Multiple functions and essential roles of nuclear receptor coactivators of bHLH-PAS family.

Classical non-peptide hormones, such as steroids, retinoids, thyroid hormones, vitamin D3 and their derivatives including prostaglandins, benzoates, oxysterols, and bile acids, are collectively designated as small lipophilic ligands, acting via binding to the nuclear receptors (NRs). The NRs form a large superfamily of transcription factors that participate virtually in every key biological process. They control various aspects of animal development, fertility, gametogenesis, and numerous metabolic pathways, and can be misregulated in many types of cancers. Their enormous functional plasticity, as transcription factors, relates in part to NR-mediated interactions with plethora of coregulatory proteins upon ligand binding to their ligand binding domains (LBD), or following covalent modification. Here, we review some general views of a specific group of NR coregulators, so-called nuclear receptor coactivators (NRCs) or steroid receptor coactivators (SRCs) and highlight some of their unique functions/roles, which are less extensively mentioned and discussed in other reviews. We also try to pinpoint few neglected moments in the cooperative action of SRCs, which may also indicate their variable roles in the hormone-independent signaling pathways.