Synthesis of a DNA-Encoded Macrocyclic Library Utilizing Intramolecular Benzimidazole Formation.

Macrocycles occupy chemical space "beyond the rule of five". They bridge traditional bioactive small molecule drugs and macromolecules and have the potential to modulate challenging targets such as PPI or proteases. Here we report an on-DNA macrocyclization reaction utilizing intramolecular benzimidazole formation. A 129-million-member macrocyclic library composed of a privileged benzimidazole core, a dipeptide sequence (natural or non-natural), and linkers of varying length and flexibility was designed and synthesized.

Physiologic Targets and Modes of Action for CBL0137, a Lead for Human African Trypanosomiasis Drug Development.

CBL0137 is a lead drug for human African trypanosomiasis, caused by Trypanosoma brucei Herein, we use a four-step strategy to 1) identify physiologic targets and 2) determine modes of molecular action of CBL0137 in the trypanosome. First, we identified fourteen CBL0137-binding proteins using affinity chromatography. Second, we developed hypotheses of molecular modes of action, using predicted functions of CBL0137-binding proteins as guides. Third, we documented effects of CBL0137 on molecular pathways in the trypanosome. Fourth, we identified physiologic targets of the drug by knocking down genes encoding CBL0137-binding proteins and comparing their molecular effects to those obtained when trypanosomes were treated with CBL0137. CBL0137-binding proteins included glycolysis enzymes (aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, phosphoglycerate kinase) and DNA-binding proteins [universal minicircle sequence binding protein 2, replication protein A1 (RPA1), replication protein A2 (RPA2)]. In chemical biology studies, CBL0137 did not reduce ATP level in the trypanosome, ruling out glycolysis enzymes as crucial targets for the drug. Thus, many CBL0137-binding proteins are not physiologic targets of the drug. CBL0137 inhibited 1) nucleus mitosis, 2) nuclear DNA replication, and 3) polypeptide synthesis as the first carbazole inhibitor of eukaryote translation. RNA interference (RNAi) against RPA1 inhibited both DNA synthesis and mitosis, whereas RPA2 knockdown inhibited mitosis, consistent with both proteins being physiologic targets of CBL0137. Principles used here to distinguish drug-binding proteins from physiologic targets of CBL0137 can be deployed with different drugs in other biologic systems. SIGNIFICANCE STATEMENT: To distinguish drug-binding proteins from physiologic targets in the African trypanosome, we devised and executed a multidisciplinary approach involving biochemical, genetic, cell, and chemical biology experiments. The strategy we employed can be used for drugs in other biological systems.

Application of l-Threonine Aldolase to on-DNA Reactions.

Enzymatic catalysis is a highly attractive approach to the DNA encoded library technology (DEL) that has not been widely explored. In this paper, we report an l-threonine aldolase (l-TA)-catalyzed on-DNA aldol reaction to form ß-hydroxy-α-amino acids, and its diastereoselectivity determination. l-TAs from three species show good on-DNA aldehyde scope and complementary stereoselectivity. The formed aldol product can be further diversified via various reactions, which demonstrates the utility of this reaction in DEL.

The Aryl Hydrocarbon Receptor is a Critical Regulator of Tissue Factor Stability and an Antithrombotic Target in Uremia.

Patients with CKD suffer high rates of thrombosis, particularly after endovascular interventions, yet few options are available to improve management and reduce thrombotic risk. We recently demonstrated that indoxyl sulfate (IS) is a potent CKD-specific prothrombotic metabolite that induces tissue factor (TF) in vascular smooth muscle cells (vSMCs), although the precise mechanism and treatment implications remain unclear. Because IS is an agonist of the aryl hydrocarbon receptor (AHR), we first examined the relationship between IS levels and AHR-inducing activity in sera of patients with ESRD. IS levels correlated significantly with both vSMC AHR activity and TF activity. Mechanistically, we demonstrated that IS activates the AHR pathway in primary human aortic vSMCs, and further, that AHR interacts directly with and stabilizes functional TF. Antagonists directly targeting AHR enhanced TF ubiquitination and degradation and suppressed thrombosis in a postinterventional model of CKD and endovascular injury. Furthermore, AHR antagonists inhibited TF in a manner dependent on circulating IS levels. In conclusion, we demonstrated that IS regulates TF stability through AHR signaling and uncovered AHR as an antithrombotic target and AHR antagonists as a novel class of antithrombotics. Together, IS and AHR have potential as uremia-specific biomarkers and targets that may be leveraged as a promising theranostic platform to better manage the elevated thrombosis rates in patients with CKD.

Repurposing strategies for tropical disease drug discovery.

Neglected tropical diseases (NTDs) and other diseases of the developing world, such as malaria, attract research investments that are disproportionately low compared to their impact on human health worldwide. Therefore, pragmatic methods for launching new drug discovery programs have emerged that repurpose existing chemical matter as new drugs or new starting points for optimization. In this Digest we describe applications of different repurposing approaches for NTDs, and provide a means by which these approaches may be differentiated from each other. These include drug repurposing, target repurposing, target class repurposing, and lead repurposing.

Evaluation of pyrrolidine and pyrazolone derivatives as inhibitors of trypanosomal phosphodiesterase B1 (TbrPDEB1).

Human African trypanosomiasis (HAT) is a parasitic disease, caused by the protozoan pathogen Trypanosoma brucei, which affects thousands every year and which is in need of new therapeutics. Herein we report the synthesis and assessment of a series of pyrrolidine and pyrazolone derivatives of human phosphodiesterase 4 (hPDE4) inhibitors for the assessment of their activity against the trypanosomal phosphodiesterase TbrPDEB1. The synthesized compounds showed weak potency against TbrPDEB1.

In silico identification of an aryl hydrocarbon receptor antagonist with biological activity in vitro and in vivo.

The aryl hydrocarbon receptor (AHR) is critically involved in several physiologic processes, including cancer progression and multiple immune system activities. We, and others, have hypothesized that AHR modulators represent an important new class of targeted therapeutics. Here, ligand shape-based virtual modeling techniques were used to identify novel AHR ligands on the basis of previously identified chemotypes. Four structurally unique compounds were identified. One lead compound, 2-((2-(5-bromofuran-2-yl)-4-oxo-4H-chromen-3-yl)oxy)acetamide (CB7993113), was further tested for its ability to block three AHR-dependent biologic activities: triple-negative breast cancer cell invasion or migration in vitro and AHR ligand-induced bone marrow toxicity in vivo. CB7993113 directly bound both murine and human AHR and inhibited polycyclic aromatic hydrocarbon (PAH)- and TCDD-induced reporter activity by 75% and 90% respectively. A novel homology model, comprehensive agonist and inhibitor titration experiments, and AHR localization studies were consistent with competitive antagonism and blockade of nuclear translocation as the primary mechanism of action. CB7993113 (IC50 3.3 × 10(-7) M) effectively reduced invasion of human breast cancer cells in three-dimensional cultures and blocked tumor cell migration in two-dimensional cultures without significantly affecting cell viability or proliferation. Finally, CB7993113 effectively inhibited the bone marrow ablative effects of 7,12-dimethylbenz[a]anthracene in vivo, demonstrating drug absorption and tissue distribution leading to pharmacological efficacy. These experiments suggest that AHR antagonists such as CB7993113 may represent a new class of targeted therapeutics for immunomodulation and/or cancer therapy.

A chemical screen identifies small molecules that regulate hepcidin expression.

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.

Repurposing human PDE4 inhibitors for neglected tropical diseases: design, synthesis and evaluation of cilomilast analogues as Trypanosoma brucei PDEB1 inhibitors.

A medicinal chemistry exploration of the human phosphodiesterase 4 (hPDE4) inhibitor cilomilast (1) was undertaken in order to identify inhibitors of phosphodiesterase B1 of Trypanosoma brucei (TbrPDEB1). T. brucei is the parasite which causes African sleeping sickness, a neglected tropical disease that affects thousands each year, and TbrPDEB1 has been shown to be an essential target of therapeutic relevance. Noting that 1 is a weak inhibitor of TbrPDEB1, we report the design and synthesis of analogs of this compound, culminating in 12b, a sub-micromolar inhibitor of TbrPDEB1 that shows modest inhibition of T. brucei proliferation.

Synthesis and assessment of catechol diether compounds as inhibitors of trypanosomal phosphodiesterase B1 (TbrPDEB1).

Human African trypanosomiasis (HAT) is a parasitic neglected tropical disease that affects 10,000 patients each year. Current treatments are sub-optimal, and the disease is fatal if not treated. Herein, we report our continuing efforts to repurpose the human phosphodiesterase 4 (hPDE4) inhibitor piclamilast to target trypanosomal phosphodiesterase TbrPDEB1. We prepared a range of substituted heterocyclic replacements for the 4-amino-3,5-dichloro-pyridine headgroup of piclamilast, and found that these compounds exhibited weak inhibitory activity of TbrPDEB1.

Chemical Optimization of CBL0137 for Human African Trypanosomiasis Lead Drug Discovery.

The carbazole CBL0137 (1) is a lead for drug development against human African trypanosomiasis (HAT), a disease caused by Trypanosoma brucei. To advance 1 as a candidate drug, we synthesized new analogs that were evaluated for the physicochemical properties, antitrypanosome potency, selectivity against human cells, metabolism in microsomes or hepatocytes, and efflux ratios. Structure-activity/property analyses of analogs revealed eight new compounds with higher or equivalent selectivity indices (5j, 5t, 5v, 5w, 5y, 8d, 13i, and 22e). Based on the overall compound profiles, compounds 5v and 5w were selected for assessment in a mouse model of HAT; while 5v demonstrated a lead-like profile for HAT drug development, 5w showed a lack of efficacy. Lessons from these studies will inform further optimization of carbazoles for HAT and other indications.

Structure-Property Optimization of a Series of Imidazopyridines for Visceral Leishmaniasis.

Leishmaniasis is a collection of diseases caused by more than 20 Leishmania parasite species that manifest as either visceral, cutaneous, or mucocutaneous leishmaniasis. Despite the significant mortality and morbidity associated with leishmaniasis, it remains a neglected tropical disease. Existing treatments have variable efficacy, significant toxicity, rising resistance, and limited oral bioavailability, which necessitates the development of novel and affordable therapeutics. Here, we report on the continued optimization of a series of imidazopyridines for visceral leishmaniasis and a scaffold hop to a series of substituted 2-(pyridin-2-yl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazoles with improved absorption, distribution, metabolism, and elimination properties.

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.

Corrigendum: Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 2. Tadalafil analogs.

In this Letter we describe our ongoing target repurposing efforts focused on discovery of inhibitors of the essential trypanosomal phosphodiesterase TbrPDEB1. This enzyme has been implicated in virulence of Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). We outline the synthesis and biological evaluation of analogs of tadalafil, a human PDE5 inhibitor currently utilized for treatment of erectile dysfunction, and report that these analogs are weak inhibitors of TbrPDEB1.

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs.

Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new therapeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phosphodiesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency analogs can be prepared, this chemical class is a sub-optimal starting point for further development of TbrPDE inhibitors.

Lead Optimization of 3,5-Disubstituted-7-Azaindoles for the Treatment of Human African Trypanosomiasis.

Neglected tropical diseases such as human African trypanosomiasis (HAT) are prevalent primarily in tropical climates and among populations living in poverty. Historically, the lack of economic incentive to develop new treatments for these diseases has meant that existing therapeutics have serious shortcomings in terms of safety, efficacy, and administration, and better therapeutics are needed. We now report a series of 3,5-disubstituted-7-azaindoles identified as growth inhibitors of Trypanosoma brucei, the parasite that causes HAT, through a high-throughput screen. We describe the hit-to-lead optimization of this series and the development and preclinical investigation of 29d, a potent antitrypanosomal compound with promising pharmacokinetic (PK) parameters. This compound was ultimately not progressed beyond in vivo PK studies due to its inability to penetrate the blood-brain barrier (BBB), critical for stage 2 HAT treatments.

Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.

This paper combines two techniques--mass spectrometry and protein charge ladders--to examine the relationship between the surface charge and hydrophobicity of a representative globular protein (bovine carbonic anhydrase II; BCA II) and its rate of amide hydrogen-deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-ε-NH3(+) groups--a type of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its secondary or tertiary structure--resulted in a linear decrease in the aggregate rate of amide H/D exchange at pD 7.4, 15 °C. According to analysis with MS, the acetylation of each additional lysine generated between 1.4 and 0.9 additional hydrogens that are protected from H/D exchange during the 2 h exchange experiment at 15 °C, pD 7.4. NMR spectroscopy demonstrated that none of the hydrogen atoms which became protected upon acetylation were located on the side chain of the acetylated lysine residues (i.e., lys-ε-NHCOCH3) but were instead located on amide NHCO moieties in the backbone. The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs of the charge ladder were the least thermostable (as measured by differential scanning calorimetry). This observation--that faster rates of exchange are associated with slower rates of denaturation--is contrary to the usual assumptions in protein chemistry. The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-ε-NHCO(CH2)2CH3]18) and peracetylated BCA II (e.g., [lys-ε-NHCOCH3]18) suggests that the electrostatic charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These electrostatic effects on the kinetics of H/D exchange could complicate (or aid) the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the protein to an oligonucleotide or to another charged ligand or protein).

Medicinal Chemistry Optimization of a Diaminopurine Chemotype: Toward a Lead for Trypanosoma brucei Inhibitors.

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei and transmitted through the bite of infected tsetse flies. The disease is considered fatal if left untreated. To identify new chemotypes against Trypanosoma brucei, previously we identified 797 potent kinase-targeting inhibitors grouped into 59 clusters plus 53 singleton compounds with at least 100-fold selectivity over HepG2 cells. From this set of hits, a cluster of diaminopurine-derived compounds was identified. Herein, we report our medicinal chemistry investigation involving the exploration of structure-activity and structure-property relationships around one of the high-throughput screening (HTS) hits, N2-(thiophen-3-yl)-N6-(2,2,2-trifluoroethyl)-9H-purine-2,6-diamine (1, NEU-1106). This work led to the identification of a potent lead compound (4aa, NEU-4854) with improved in vitro absorption, distribution, metabolism, and excretion (ADME) properties, which was progressed into proof-of-concept translation of in vitro antiparasitic activity to in vivo efficacy.