Drugging the microbiome: targeting small microbiome molecules.

The human microbiome represents a large and diverse collection of microbes that plays an integral role in human physiology and pathophysiology through interactions with the host and within the microbial community. While early work exploring links between microbiome signatures and diseases states has been associative, emerging evidence demonstrates the metabolic products of the human microbiome have more proximal causal effects on disease phenotypes. The therapeutic implications of this shift are profound as manipulation of the microbiome by the administration of live biotherapeutics, ongoing, can now be pursued alongside research efforts toward describing inhibitors of key microbiome enzymes involved in the biosynthesis of metabolites implicated in various disease states and processing of host-derived metabolites. With growing interest in 'drugging the microbiome', we review few notable microbial metabolites for which traditional drug-development campaigns have yielded compounds with therapeutic promise.

1'-Cyano Intermediate Enables Rapid and Stereoretentive Access to 1'-Modified Remdesivir Nucleosides.

While biochemical, structural, and computational studies have shown the importance of remdesivir's C1'-substituent in its perturbation of SARS-CoV-2 RdRp action, we recognized the paucity of methods to stereoselectively install substituents at this position as an obstacle to rigorous explorations of SAR and mechanism. We report the utilization of an anomerically pure 1'-cyano intermediate as an entry point to a chemically diverse set of substitutions, allowing for 1'diversification while obviating the need for the tedious separation of anomeric mixtures.

Innovative Strategies for the Construction of Diverse 1'-Modified C-Nucleoside Derivatives.

Modified C-nucleosides have proven to be enormously successful as chemical probes to understand fundamental biological processes and as small-molecule drugs for cancer and infectious diseases. Historically, the modification of the glycosyl unit has focused on the 2'-, 3'-, and 4'-positions as well as the ribofuranosyl ring oxygen. By contrast, the 1'-position has rarely been studied due to the labile nature of the anomeric position. However, the improved chemical stability of C-nucleosides allows the modification of the 1'-position with substituents not found in conventional N-nucleosides. Herein, we disclose new chemistry for the installation of diverse substituents at the 1'-position of C-nucleosides, including alkyl, alkenyl, difluoromethyl, and fluoromethyl substituents, using the 4-amino-7-(1'-hydroxy-d-ribofuranosyl)pyrrolo[2,1-f][1,2,4]triazine scaffold as a representative purine nucleoside mimetic.

4,5-Disubstituted 1,2,3-triazoles: Effective Inhibition of Indoleamine 2,3-Dioxygenase 1 Enzyme Regulates T cell Activity and Mitigates Tumor Growth.

The improvement of body's own immune system is considered one of the safest approaches to fight against cancer and several other diseases. Excessive catabolism of the essential amino acid, L-tryptophan (L-Trp) assists the cancer cells to escape normal immune obliteration. The formation of disproportionate kynurenine and other downstream metabolites suppress the T cell functions. Blocking of this immunosuppressive mechanism is considered as a promising approach against cancer, neurological disorders, autoimmunity, and other immune-mediated diseases. Overexpression of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme is directly related to the induction of immunosuppressive mechanisms and represents an important therapeutic target. Several classes of small molecule-based IDO1 inhibitors have been already reported, but only few compounds are currently being evaluated in various stages of clinical trials as adjuvants or in combination with chemo- and radiotherapies. In the quest for novel structural class(s) of IDO1 inhibitors, we developed a series of 4,5-disubstituted 1,2,3-triazole derivatives. The optimization of 4,5-disubstituted 1,2,3-triazole scaffold and comprehensive biochemical and biophysical studies led to the identification of compounds, 3i, 4i, and 4k as potent and selective inhibitors of IDO1 enzyme with IC50 values at a low nanomolar level. These potent compounds also showed strong IDO1 inhibitory activities in MDA-MB-231 cells with no/negligible level of cytotoxicity. The T cell activity studies revealed that controlled regulation of IDO1 enzyme activity in the presence of these potent compounds could induce immune response against breast cancer cells. The compounds also showed excellent in vivo antitumor efficacy (of tumor growth inhibition = 79-96%) in the female Swiss albino mice. As a consequence, this study describes the first example of 4,5-disubstituted 1,2,3-triazole based IDO1 inhibitors with potential applications for immunotherapeutic studies.

Ring-Opening of Indoles: An Unconventional Route for the Transformation of Indoles to 1 H-Pyrazoles Using Lewis Acid.

An unusual transformation of indoles to pyrazoles via an aromatic ring-opening strategy has been developed. The salient feature of this strategy involves the C2-N1 bond opening and concomitant cyclization reaction of the C2═C3 bond of the indole moiety with the tosylhydrazone, which proceeds under transition-metal and ligand free conditions. This ring-opening functionalization of indoles provides a wide scope of differently substituted pyrazoles.

Diphenylethylenediamine-Based Potent Anionophores: Transmembrane Chloride Ion Transport and Apoptosis Inducing Activities.

Synthetic anion transporters have been recognized as one of the potential therapeutic agents for the treatment of diseases including cystic fibrosis, myotonia, and epilepsy that originate due to the malfunctioning of natural Cl- ion transport systems. Recent studies showed that the synthetic Cl- ion transporters can also disrupt cellular ion-homeostasis and induce apoptosis in cancer cell lines, leading to a revived attention for synthetic Cl- ion transporters. Herein, we report the development of conformationally controlled 1,2-diphenylethylenediamine-based bis(thiourea) derivatives as a new class of selective Cl- ion carrier. The strong Cl- ion binding properties ( Kd = 3.87-6.66 mM) of the bis(thiourea) derivatives of diamine-based compounds correlate well with their transmembrane anion transport activities (EC50 = 2.09-4.15 nM). The transport of Cl- ions via Cl-/NO3- antiport mechanism was confirmed for the most active molecule. Perturbation of Cl- ion homeostasis by this anion carrier induces cell death by promoting the caspase-mediated intrinsic pathway of apoptosis.

Azidophosphonate Chemistry as a Route for a Novel Class of Vesicle-Forming Phosphonolipids.

Membrane forming synthetic lipids constitutes a new class of biomaterials with impressive applications in the field of biological and pharmaceutical sciences. Interestingly, alteration(s) in the headgroup region of the lipids offer a wide chemical space to investigate their specific properties. In this regard, we have utilized ß-azidophosphonate chemistry to gain access to a novel class of triazole-phosphonate (TP) amphiphiles with fascinating physicochemical properties of lipids. TP lipids form stable vesicles that exhibit negative surface potential across a broad pH range. These anionic lipids have high phase-transition temperatures, phospholipase resistance, slow vesicle leakage profiles, and doxorubicin delivery efficacy. We hypothesize that these readily synthesizable phosphonolipids could find several applications as phospholipid substituents.

Transition Metal, Azide, and Oxidant-Free Homo- and Heterocoupling of Ambiphilic Tosylhydrazones to the Regioselective Triazoles and Pyrazoles.

With N-tosylhydrazone as an ambiphilic reagent, an unprecedented cyclization reaction of two identical or different tosylhydrazones has been developed to access various 4,5-disubstituted-2H-triazoles under transition metal, azide, and oxidant-free conditions. A mechanistic rationalization study led to the identification of several electronically diverse unsaturated systems for regioselective synthesis of 1- and 2-substituted 1,2,3-triazoles and pyrazoles.

Synthesis and evaluation of oxindoles as promising inhibitors of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is considered as an important therapeutic target for the treatment of cancer, chronic infections and other diseases that are associated with immune suppression. Recent developments in understanding the catalytic mechanism of the IDO1 enzyme revealed that conversion of l-tryptophan (l-Trp) to N-formylkynurenine proceeded through an epoxide intermediate state. Accordingly, we synthesized a series of 3-substituted oxindoles from l-Trp, tryptamine and isatin. Compounds with C3-substituted oxindole moieties showed moderate inhibitory activity against the purified human IDO1 enzyme. Their optimization led to the identification of potent compounds, 6, 22, 23 and 25 (IC50 = 0.19 to 0.62 µM), which are competitive inhibitors of IDO1 with respect to l-Trp. These potent compounds also showed IDO1 inhibition potencies in the low-micromolar range (IC50 = 0.33-0.49 µM) in MDA-MB-231 cells. The cytotoxicity of these potent compounds was trivial in different model cancer (MDA-MB-231, A549 and HeLa) cells and macrophage (J774A.1) cells. Stronger selectivity for the IDO1 enzyme (124 to 210-fold) over the tryptophan 2,3-dioxygenase (TDO) enzyme was also observed for these compounds. These results suggest that the oxindole moiety of the compounds could mimic the epoxide intermediate state of l-Trp. Therefore, the structural simplicity and low-micromolar inhibition potencies of these 3-substituted oxindoles make them quite attractive for further investigation of IDO1 function and immunotherapeutic applications.

Fused Heterocyclic Compounds as Potent Indoleamine-2,3-dioxygenase 1 Inhibitors.

Uncontrolled metabolism of l-tryptophan (l-Trp) in the immune system has been recognized as a critical cellular process in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) enzyme plays an important role in the metabolism of a local l-Trp through the kynurenine pathway in the immune systems. In this regard, IDO1 has emerged as a therapeutic target for the treatment of diseases that are associated with immune suppression like chronic infections, cancer, and others. In this study, we synthesized a series of pyridopyrimidine, pyrazolopyranopyrimidine, and dipyrazolopyran derivatives. Further lead optimizations directed to the identification of potent compounds, 4j and 4l (IC50 = 260 and 151 nM, respectively). These compounds also exhibited IDO1 inhibitory activities in the low nanomolar range in MDA-MB-231 cells with very low cytotoxicity. Stronger selectivity for the IDO1 enzyme (>300-fold) over tryptophan 2,3-dioxygenase (TDO) enzyme was also observed for these compounds. Hence, these fused heterocyclic compounds are attractive candidates for the advanced study of IDO1-dependent cellular function and immunotherapeutic applications.

Phosphate bioisostere containing amphiphiles: a novel class of squaramide-based lipids.

We describe a novel class of amphiphiles with squaramide moiety as a phosphate bioisostere. Most synthesized squaramide-based amphiphiles have the favorable physicochemical properties of lipids, such as: formation of stable liposomes or giant unilamellar vesicles in aqueous solution, high phase-transition temperature, low vesicle leakage and phospholipase resistance properties.

Nitrobenzofurazan derivatives of N'-hydroxyamidines as potent inhibitors of indoleamine-2,3-dioxygenase 1.

Tryptophan metabolism through the kynurenine pathway is considered as a crucial mechanism in immune tolerance. Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tryptophan catabolism in the immune system and it is also considered as an important therapeutic target for the treatment of cancer and other diseases that are linked with kynurenine pathway. In this study, a series of nitrobenzofurazan derivatives of N'-hydroxybenzimidamides (1) and N'-hydroxy-2-phenylacetimidamides (2) were synthesized and their inhibitory activities against human IDO1 enzyme were tested using in-vitro and cellular enzyme activity assay. The optimization leads to the identification of potent compounds, 1d, 2i and 2k (IC50 = 39-80 nM), which are either competitive or uncompetitive inhibitors of IDO1 enzyme. These compounds also showed IDO1 inhibition potencies in the nanomolar range (IC50 = 50-71 nM) in MDA-MB-231 cells with no/negligible amount of cytotoxicity. The stronger selectivity of the potent compounds for IDO1 enzyme over tryptophan 2,3-dioxygenase (TDO) enzyme (312-1593-fold) also makes them very attractive for further immunotherapeutic applications.

Chemistry of N,S-Heterocyclic Carbene and Metallaboratrane Complexes: A New η(3)-BCC-Borataallyl Complex.

A high-yielding synthetic route for the preparation of group 9 metallaboratrane complexes [Cp*MBH(L)2], 1 and 2 (1, M=Rh, 2, M=Ir; L=C7H4NS2) has been developed using [{Cp*MCl2}2] as precursor. This method also permitted the synthesis of an Rh-N,S-heterocyclic carbene complex, [(Cp*Rh)(L2)(1-benzothiazol-2-ylidene)] (3; L=C7H4NS2) in good yield. The reaction of compound 3 with neutral borane reagents led to the isolation of a novel borataallyl complex [Cp*Rh(L)2B{CH2C(CO2Me)}] (4; L=C7H4NS2). Compound 4 features a rare η(3)-interaction between rhodium and the B-C-C unit of a vinylborane moiety. Furthermore, with the objective of generating metallaboratranes of other early and late transition metals through a transmetallation approach, reactions of rhoda- and irida-boratrane complexes with metal carbonyl compounds were carried out. Although the objective of isolating such complexes was not achieved, several interesting mixed-metal complexes [{Cp*Rh}{Re(CO)3}(C7H4NS2)3] (5), [Cp*Rh{Fe2(CO)6}(µ-CO)S] (6), and [Cp*RhBH(L)2W(CO)5] (7; L=C7H4NS2) have been isolated. All of the new compounds have been characterized in solution by mass spectrometry, IR spectroscopy, and (1)H, (11)B, and (13)C NMR spectroscopies, and the structural types of 4-7 have been unequivocally established by crystallographic analysis.

Elucidating the interaction of γ-hydroxymethyl-γ-butyrolactone substituents with model membranes and protein kinase C-C1 domains.

The protein kinase C (PKC) family of proteins is an attractive drug target. Dysregulation of PKC-dependent signalling pathways is related to several human diseases like cancer, immunological and other diseases. We approached the problem of altering PKC activities by developing C1 domain-based PKC ligands. In this report γ-hydroxymethyl-γ-butyrolactone (HGL) substituents were investigated in an effort to develop small molecule-based PKC regulators with higher specificity for C1 domain than the endogenous diacylglycerols (DAGs). Extensive analysis of membrane-ligands interaction measurements revealed that the membrane-active compounds strongly interact with the lipid bilayers and the hydrophilic parts of compounds localize at the bilayer/water interface. The pharmacophores like hydroxymethyl, carbonyl groups and acyl-chain length of the compounds are crucial for their interaction with the C1 domain proteins. The potent compounds showed more than 17-fold stronger binding affinity for the C1 domains than DAG under similar experimental conditions. Nonradioactive kinase assay confirmed that these potent compounds have similar or better PKC dependent phosphorylation capabilities than DAG under similar experimental conditions. Hence, our findings reveal that these HGL analogues represent an attractive group of structurally simple C1 domain ligands that can be further structurally altered to improve their potencies.

Synthesis and protein kinase C (PKC)-C1 domain binding properties of diacyltetrol based anionic lipids.

The protein kinase C (PKC) family of lipid-activated kinases plays a significant role in the regulation of diverse cellular functions including tumor promotion, apoptosis, differentiation, and others. The lipophilic second messenger diacylglycerols (DAGs) act as endogenous ligands for the PKCs in the presence of anionic phospholipids. To develop effective PKC regulators and understand the importance of anionic phospholipids in DAG binding of PKC isoforms, we conveniently synthesized octanoic acid containing diacyltetrol (DAT) based hybrid lipids with both DAG and anionic phospholipid headgroups within the same molecule. We also used palmitic and oleic acid containing hybrid lipids for additional understanding of the PKC-C1 domain binding mechanism. Biophysical studies showed that hydrophobic side chains, DAG and anionic phospholipids headgroups are necessary for their interaction with the C1-domain of PKC isoforms. The hybrid lipids DAT-PS and DAT-PA specifically interact with the PKCδ-C1b and PKCθ-C1b subdomains and showed 5- and 2.5-fold stronger binding affinity compared with DAG, respectively. Whereas, the PKCα-C1a subdomain interacts with the hybrid lipids, without any significant specificity. The present results show that hybrid lipids bind to the PKC C1b/a subdomains and can be further studied to decipher their binding mechanism and biological activities. This study proposes a new concept of developing PKC activators by using tetrol-based anionic hybrid lipids having both phospholipids and diacylglycerol headgroups within the same molecule. This study also supplies useful information for the binding potencies of hybrid lipids with PKC-C1 domains.

Membrane Interaction and Protein Kinase C-C1 Domain Binding Properties of 4-Hydroxy-3-(hydroxymethyl) Phenyl Ester Analogues.

Protein kinase C (PKC)-C1 domain targeted regulator development is considered as a potential therapeutic strategy for the treatment of cancer and immunological and other diseases. Efforts are underway to synthesize small molecules to achieve higher specificity for the C1-domain than the natural activator, diacylglycerols (DAGs). In this regard, we conveniently synthesized 4-hydroxy-3-(hydroxymethyl) phenyl ester analogues and measured in vitro C1-domain binding properties. We also investigated different physicochemical properties of the synthesized molecules, including aggregation behavior in aqueous solution and interaction with lipid bilayers, and others with an aim for better understanding of their C1-domain binding properties. The results showed that the membrane-active compounds aggregate in aqueous solution at a reasonably lower concentration and strongly interact with the lipid bilayer. The hydrophilic part of the compounds localize at the bilayer/water interface and accessible for C1-domain binding. Biophysical studies revealed that the hydroxyl, hydroxymethyl, and carbonyl groups and acyl chain length are important for their interaction with the C1-domain. The potent compound showed more than 10-fold stronger binding affinity for the C1-domains than DAG under similar experimental conditions. Therefore, our findings reveal that these ester analogues represent an attractive group of C1-domain ligands that can be further structurally modified to improve their binding and activity.