Thiophenyl Derivatives of Nicotinamide Are Metabolized by the NAD Salvage Pathway into Unnatural NAD Derivatives That Inhibit IMPDH and Are Toxic to Peripheral Nerve Cancers.

N-Pyridinylthiophene carboxamide (compound 21) displays activity against peripheral nerve sheath cancer cells and mouse xenografts by an unknown mechanism. Through medicinal chemistry, we identified a more active derivative, compound 9, and found that only analogues with structures similar to nicotinamide retained activity. Genetic screens using compound 9 found that both NAMPT and NMNAT1, enzymes in the NAD salvage pathway, are necessary for activity. Compound 9 is metabolized by NAMPT and NMNAT1 into an adenine dinucleotide (AD) derivative in a cell-free system, cultured cells, and mice, and inhibition of this metabolism blocked compound activity. AD analogues derived from compound 9 inhibit IMPDH in vitro and cause cell death by inhibiting IMPDH in cells. These findings nominate these compounds as preclinical candidates for the development of tumor-activated IMPDH inhibitors to treat neuronal cancers.

Chemoselective and Stereoselective Allylation of Bis(alkenyl)boronates.

Bis(alkenyl)boronates react with optically active Ir(π-allyl) species in a process that involves allylation of the more substituted olefin and 1,2-metalate shift of the less substituted olefin. The method constructs valuable enantioenriched tertiary allylic boronic esters with high chemoselectivity, enantioselectivity and diastereoselectivity. Allylic functionalization reactions transform the 1,3-stereodiad to 1,5- and 1,6-stereochemical relationships.

Pseudomonas effector AvrB is a glycosyltransferase that rhamnosylates plant guardee protein RIN4.

The plant pathogen Pseudomonas syringae encodes a type III secretion system avirulence effector protein, AvrB, that induces a form of programmed cell death called the hypersensitive response in plants as a defense mechanism against systemic infection. Despite the well-documented catalytic activities observed in other Fido (Fic, Doc, and AvrB) proteins, the enzymatic activity and target substrates of AvrB have remained elusive. Here, we show that AvrB is an unprecedented glycosyltransferase that transfers rhamnose from UDP-rhamnose to a threonine residue of the Arabidopsis guardee protein RIN4. We report structures of various enzymatic states of the AvrB-catalyzed rhamnosylation reaction of RIN4, which reveal the structural and mechanistic basis for rhamnosylation by a Fido protein. Collectively, our results uncover an unexpected reaction performed by a prototypical member of the Fido superfamily while providing important insights into the plant hypersensitive response pathway and foreshadowing more diverse chemistry used by Fido proteins and their substrates.

Enzymatic Resolution and Decarboxylative Functionalization of α-Sulfinyl Esters.

α-Sulfinyl esters can be readily prepared through thiol substitution of α-bromo esters followed by oxidation to the sulfoxide. Enzymatic resolution with lipoprotein lipase provides both the unreacted esters and corresponding α-sulfinyl carboxylic acids in high yields and enantiomeric ratios. Subsequent decarboxylative halogenation, dihalogenation, trihalogenation and cross-coupling gives rise to functionalized sulfoxides. The method has been applied to the asymmetric synthesis of a potent inhibitor of 15-prostaglandin dehydrogenase.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Small molecule inhibitors of 15-PGDH exploit a physiologic induced-fit closing system.

15-prostaglandin dehydrogenase (15-PGDH) is a negative regulator of tissue stem cells that acts via enzymatic activity of oxidizing and degrading PGE2, and related eicosanoids, that support stem cells during tissue repair. Indeed, inhibiting 15-PGDH markedly accelerates tissue repair in multiple organs. Here we have used cryo-electron microscopy to solve the solution structure of native 15-PGDH and of 15-PGDH individually complexed with two distinct chemical inhibitors. These structures identify key 15-PGDH residues that mediate binding to both classes of inhibitors. Moreover, we identify a dynamic 15-PGDH lid domain that closes around the inhibitors, and that is likely fundamental to the physiologic 15-PGDH enzymatic mechanism. We furthermore identify two key residues, F185 and Y217, that act as hinges to regulate lid closing, and which both inhibitors exploit to capture the lid in the closed conformation, thus explaining their sub-nanomolar binding affinities. These findings provide the basis for further development of 15-PGDH targeted drugs as therapeutics for regenerative medicine.

Diastereoselective Alkylation of Activated Nitrogen Heterocycles with Alkenyl Boronate Complexes.

Alkenyl boronate complexes react with acylated quinolines and isoquinolines via 1,2-metalate rearrangement to give alkylated, dearomatized heterocycles in good yields, diastereoselectivities, and regioselectivities. This multi-component coupling is highly modular and can be used to access a wide scope of heterocyclic scaffolds. Chiral boronic esters made through this methodology possess high synthetic potential and can be transformed into various functional groups in one step without racemization.

Fast-Killing Tyrosine Amide ((S)-SW228703) with Blood- and Liver-Stage Antimalarial Activity Associated with the Cyclic Amine Resistance Locus (PfCARL).

Current malaria treatments are threatened by drug resistance, and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) and P. falciparum acetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their targets are unknown. The tyramine of (S)-SW703 is shared with some reported PfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for the activity of (S)-SW703, suggesting differences in the mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity for multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.

Orally Bioavailable Quinoxaline Inhibitors of 15-Prostaglandin Dehydrogenase (15-PGDH) Promote Tissue Repair and Regeneration.

15-Prostaglandin dehydrogenase (15-PGDH) regulates the concentration of prostaglandin E2 in vivo. Inhibitors of 15-PGDH elevate PGE2 levels and promote tissue repair and regeneration. Here, we describe a novel class of quinoxaline amides that show potent inhibition of 15-PGDH, good oral bioavailability, and protective activity in mouse models of ulcerative colitis and recovery from bone marrow transplantation.

Mechanistic Basis for the Iridium-Catalyzed Enantioselective Allylation of Alkenyl Boronates.

Iridium(phosphoramidite) complexes catalyze an enantio- and diastereoselective three-component coupling reaction of alkenyl boronic esters, organolithium reagents, and secondary allylic carbonates. The reaction proceeds through an allylation-induced 1,2-metalate shift of the alkenyl boronate to form non-adjacent stereocenters. Mechanistic investigations outline the overall catalytic cycle and reveal trends in reactivity and selectivity. Analysis of relative stereochemistry in products derived from a variety of 1,1-disubtituted alkenyl boronates provides insight into the transition state of the addition and indicates a concerted pathway. Kinetic analysis of the reaction revealed the kinetic order dependence in boronate, the catalyst, and both the slow- and fast-reacting enantiomer of allylic carbonate as well as the turnover-limiting step of the reaction. Determination of nucleophile-specific parameters N and sN for alkenyl boronate complexes enabled comparison to other classes of nucleophiles. DFT calculations indicate the addition of the alkenyl boronate to the cationic Ir(π-allyl) intermediate and the 1,2-metalate shift occur in a concerted mechanism. The stereoselectivity is determined by ligand-substrate steric repulsions and dispersion interactions in the syn addition transition state. Hammett studies supported the computational results with regard to electronic trends observed with both aryl-derived alkenyl boronates and aryl carbonates.

A male-derived nonribosomal peptide pheromone controls female schistosome development.

Schistosomes cause morbidity and death throughout the developing world due to the massive numbers of eggs female worms deposit into the blood of their host. Studies dating back to the 1920s show that female schistosomes rely on constant physical contact with a male worm both to become and remain sexually mature; however, the molecular details governing this process remain elusive. Here, we uncover a nonribosomal peptide synthetase that is induced in male worms upon pairing with a female and find that it is essential for the ability of male worms to stimulate female development. We demonstrate that this enzyme generates ß-alanyl-tryptamine that is released by paired male worms. Furthermore, synthetic ß-alanyl-tryptamine can replace male worms to stimulate female sexual development and egg laying. These data reveal that peptide-based pheromone signaling controls female schistosome sexual maturation, suggesting avenues for therapeutic intervention and uncovering a role for nonribosomal peptides as metazoan signaling molecules.

In vivo CRISPR screening identifies BAZ2 chromatin remodelers as druggable regulators of mammalian liver regeneration.

Identifying new pathways that regulate mammalian regeneration is challenging due to the paucity of in vivo screening approaches. We employed pooled CRISPR knockout and activation screening in the regenerating liver to evaluate 165 chromatin regulatory proteins. Both screens identified the imitation-SWI chromatin remodeling components Baz2a and Baz2b, not previously implicated in regeneration. In vivo sgRNA, siRNA, and knockout strategies against either paralog confirmed increased regeneration. Distinct BAZ2-specific bromodomain inhibitors, GSK2801 and BAZ2-ICR, resulted in accelerated liver healing after diverse injuries. Inhibitor-treated mice also exhibited improved healing in an inflammatory bowel disease model, suggesting multi-tissue applicability. Transcriptomics on regenerating livers showed increases in ribosomal and cell cycle mRNAs. Surprisingly, CRISPRa screening to define mechanisms showed that overproducing Rpl10a or Rpl24 was sufficient to drive regeneration, whereas Rpl24 haploinsufficiency was rate limiting for BAZ2 inhibition-mediated regeneration. The discovery of regenerative roles for imitation-SWI components provides immediate strategies to enhance tissue repair.

Toward a Best-in-Class Inhibitor of Cholesteryl Ester Transfer Protein (CETP).

Inhibitors of cholesteryl ester transfer protein (CETP) elevate HDL levels human clinical trials. However, the first CETP inhibitors proved toxic in pivotal trials or showed minimal therapeutic benefit. Anacetrapib showed some clinical benefit but is high lipophilic. This Viewpoint highlights efforts to optimize anacetrapib to a best-in-class CETP inhibitor.

Enantioselective Allylation of Alkenyl Boronates Promotes a 1,2-Metalate Rearrangement with 1,3-Diastereocontrol.

Alkenyl boronates add to Ir(π-allyl) intermediates with high enantioselectivity. A 1,2-metalate shift forms a second C-C bond and sets a 1,3-stereochemical relationship. The three-component coupling provides tertiary boronic esters that can undergo multiple additional functionalizations. An extension to trisubstituted olefins sets three contiguous stereocenters.

15-PGDH inhibition activates the splenic niche to promote hematopoietic regeneration.

The splenic microenvironment regulates hematopoietic stem and progenitor cell (HSPC) function, particularly during demand-adapted hematopoiesis; however, practical strategies to enhance splenic support of transplanted HSPCs have proved elusive. We have previously demonstrated that inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH), using the small molecule (+)SW033291 (PGDHi), increases BM prostaglandin E2 (PGE2) levels, expands HSPC numbers, and accelerates hematologic reconstitution after BM transplantation (BMT) in mice. Here we demonstrate that the splenic microenvironment, specifically 15-PGDH high-expressing macrophages, megakaryocytes (MKs), and mast cells (MCs), regulates steady-state hematopoiesis and potentiates recovery after BMT. Notably, PGDHi-induced neutrophil, platelet, and HSPC recovery were highly attenuated in splenectomized mice. PGDHi induced nonpathologic splenic extramedullary hematopoiesis at steady state, and pretransplant PGDHi enhanced the homing of transplanted cells to the spleen. 15-PGDH enzymatic activity localized specifically to macrophages, MK lineage cells, and MCs, identifying these cell types as likely coordinating the impact of PGDHi on splenic HSPCs. These findings suggest that 15-PGDH expression marks HSC niche cell types that regulate hematopoietic regeneration. Therefore, PGDHi provides a well-tolerated strategy to therapeutically target multiple HSC niches, promote hematopoietic regeneration, and improve clinical outcomes of BMT.

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

Hydroesterification and difunctionalization of olefins with N-hydroxyphthalimide esters.

Irradiation of aryl esters of N-hydroxyphthalimides in the presence of unactivated olefins promotes a mild and regioselective hydroesterification. Optimal results are obtained with the aid of fac-Ir(dFppy)3 in CH2Cl2. Terminal and 1,1-disubstituted olefins provide primary esters, and trisubstituted olefins provide secondary esters. The anti-Markovnikov selectivity is consistent with alkyl radical intermediates, which are also indicated by the formation of cyclized products from dienes. Mono-acylated diols are formed from tri- and tetrasubstituted olefins in the presence of water.

Tumor-Activated Benzothiazole Inhibitors of Stearoyl-CoA Desaturase.

A series of N-acyl benzothiazoles shows selective and potent cytotoxicity against cancer cell lines expressing cytochrome P450 4F11. A prodrug form is metabolized by cancer cells into an active inhibitor of stearoyl-CoA desaturase (SCD). Substantial variation on the acyl portion of the inhibitors allowed the identification of (R)-27, which balanced potency, solubility, and lipophilicity to allow proof-of-concept studies in mice. The prodrugs were activated inside the tumor, where they can arrest tumor growth. Together, these observations offer promise that a tumor-activated prodrug strategy might exploit the essentiality of SCD for tumor growth, while avoiding toxicity associated with systemic SCD inhibition.

An Antiparasitic Compound from the Medicines for Malaria Venture Pathogen Box Promotes Leishmania Tubulin Polymerization.

The few frontline antileishmanial drugs are poorly effective and toxic. To search for new drugs for this neglected tropical disease, we tested the activity of compounds in the Medicines for Malaria Venture (MMV) "Pathogen Box" against Leishmania amazonensis axenic amastigotes. Screening yielded six discovery antileishmanial compounds with EC50 values from 50 to 480 nM. Concentration-response assays demonstrated that the best hit, MMV676477, had mid-nanomolar cytocidal potency against intracellular Leishmania amastigotes, Trypanosoma brucei, and Plasmodium falciparum, suggesting broad antiparasitic activity. We explored structure-activity relationships (SAR) within a small group of MMV676477 analogs and observed a wide potency range (20-5000 nM) against axenic Leishmania amastigotes. Compared to MMV676477, our most potent analog, SW41, had ∼5-fold improved antileishmanial potency. Multiple lines of evidence suggest that MMV676477 selectively disrupts Leishmania tubulin dynamics. Morphological studies indicated that MMV676477 and analogs affected L. amazonensis during cell division. Differential centrifugation showed that MMV676477 promoted partitioning of cellular tubulin toward the polymeric form in parasites. Turbidity assays with purified Leishmania and porcine tubulin demonstrated that MMV676477 promoted leishmanial tubulin polymerization in a concentration-dependent manner. Analogs' antiparasitic activity correlated with their ability to facilitate purified Leishmania tubulin polymerization. Chemical cross-linking demonstrated binding of the MMV676477 scaffold to purified Leishmania tubulin, and competition studies established a correlation between binding and antileishmanial activity. Our studies demonstrate that MMV676477 is a potent antiparasitic compound that preferentially promotes Leishmania microtubule polymerization. Due to its selectivity for and broad-spectrum activity against multiple parasites, this scaffold shows promise for antiparasitic drug development.