Chemical proteomic identification of functional cysteines with atypical electrophile reactivities.

Cysteine-directed covalent ligands have emerged as a versatile category of chemical probes and drugs that leverage thiol nucleophilicity to form permanent adducts with proteins of interest. Understanding the scope of cysteines that can be targeted by covalent ligands, as well as the types of electrophiles that engage these residues, represent important challenges for fully realizing the potential of cysteine-directed chemical probe discovery. Although chemical proteomic strategies have begun to address these important questions, only a limited number of electrophilic chemotypes have been explored to date. Here, we describe a diverse set of candidate electrophiles appended to a common core 6-methoxy-1,2,3,4-tetrahydroquinoline fragment and evaluate their global cysteine reactivity profiles in human cancer cell proteomes. This work uncovered atypical reactivity patterns for a discrete set of cysteines, including residues involved in enzymatic catalysis and located in proximity to protein-protein interactions. These findings thus point to potentially preferred electrophilic groups for site-selectively targeting functional cysteines in the human proteome.

The quest for supernatural products: the impact of total synthesis in complex natural products medicinal chemistry.

Covering: 2000 up to 2020This review presents select recent advances in the medicinal chemistry of complex natural products that are prepared by total synthesis. The underlying studies highlight enabling divergent synthetic strategies and methods that permit the systematic medicinal chemistry studies of key analogues bearing deep-seated structural changes not readily accessible by semisynthetic or biosynthetic means. Select and recent examples are detailed where the key structural changes are designed to improve defined properties or to overcome an intrinsic limitation of the natural product itself. In the examples presented, the synthetic efforts provided supernatural products, a term first introduced by our colleague Ryan Shenvi (Synlett, 2016, 27, 1145-1164), with properties superseding the parent natural product. The design principles and approaches for creating the supernatural products are highlighted with an emphasis on the properties addressed that include those that improve activity or potency, increase selectivity, enhance durability, broaden the spectrum of activity, improve chemical or metabolic stability, overcome limiting physical properties, add mechanisms of action, enhance PK properties, overcome drug resistance, and/or improve in vivo efficacy. Some such improvements may be regarded by some as iterative enhancements whereas others, we believe, truly live up to their characterization as supernatural products. Most such efforts are also accompanied by advances in synthetic organic chemistry, inspiring the development of new synthetic methodology and providing supernatural products with improved synthetic accessibility.

SAR studies of 4-acyl-1,6-dialkylpiperazin-2-one arenavirus cell entry inhibitors.

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

Inverse Electron Demand Diels-Alder Reactions of Heterocyclic Azadienes, 1-Aza-1,3-Butadienes, Cyclopropenone Ketals, and Related Systems. A Retrospective.

A summary of the investigation and applications of the inverse electron demand Diels-Alder reaction is provided that have been conducted in our laboratory over a period that now spans more than 35 years. The work, which continues to provide solutions to complex synthetic challenges, is presented in the context of more than 70 natural product total syntheses in which the reactions served as a key strategic step in the approach. The studies include the development and use of the cycloaddition reactions of heterocyclic azadienes (1,2,4,5-tetrazines; 1,2,4-, 1,3,5-, and 1,2,3-triazines; 1,2-diazines; and 1,3,4-oxadiazoles), 1-aza-1,3-butadienes, α-pyrones, and cyclopropenone ketals. Their applications illustrate the power of the methodology, often provided concise and nonobvious total syntheses of the targeted natural products, typically were extended to the synthesis of analogues that contain deep-seated structural changes in more comprehensive studies to explore or optimize their biological properties, and highlight a wealth of opportunities not yet tapped.

Ultra-potent vinblastine analogues improve on-target activity of the parent microtubulin-targeting compound.

In recent efforts, several C20' urea vinblastine analogues were discovered that displayed remarkable potency against vinblastine-sensitive tumor cell lines (IC50 50-75 pM), being roughly 100-fold more potent than vinblastine, and that exhibited decreased susceptibility to Pgp efflux-derived resistance in a vinblastine-resistant cell line. Their extraordinary activity indicate that it is not likely or even possible that their cellular functional activity is derived from stoichiometric occupancy of the intracellular tubulin binding sites. Rather, their potency indicates sub-stoichiometric or even catalytic occupancy of candidate binding sites may be sufficient to disrupt tubulin dynamics or microtubule assembly during mitosis. We detail efforts to delineate the underlying behavior responsible for the increased potency and show that the ultra-potent extended C20' ureas retain the mechanistic behavior of vinblastine, display enhanced affinity for tubulin and on-target activity approximately 100-fold both in vitro and in HeLa cells, but do not show evidence of catalytic disassembly of microtubulin. We also use the analogues to show that, in live interphase cells, the effects of the vinblastine class of drugs do not display a catastrophic effect on the microtubule skeleton, but rather a subtler insult to its dynamicity, acting as sub-stoichiometric drugs that inhibit normal microtubulin maturation and dynamics.

Structural Basis of TLR2/TLR1 Activation by the Synthetic Agonist Diprovocim.

Diprovocim is a recently discovered exceptionally potent, synthetic small molecule agonist of TLR2/TLR1 and has shown significant adjuvant activity in anticancer vaccination against murine melanoma. Since Diprovocim bears no structural similarity to the canonical lipopeptide ligands of TLR2/TLR1, we investigated how Diprovocim interacts with TLR2/TLR1 through in vitro biophysical, structural, and computational approaches. We found that Diprovocim induced the formation of TLR2/TLR1 heterodimers as well as TLR2 homodimers in vitro. We determined the crystal structure of Diprovocim in a complex with a TLR2 ectodomain, which revealed, unexpectedly, two Diprovocim molecules bound to the ligand binding pocket formed between two TLR2 ectodomains. Extensive hydrophobic interactions and a hydrogen-bonding network between the protein and Diprovocim molecules are observed within the defined ligand binding pocket and likely underlie the high potency of Diprovocim. Our work shed first light into the activation mechanism of TLR2/TLR1 by a noncanonical agonist. The structural information obtained here may be exploited to manipulate TLR2/TLR1-dependent signaling.

A small molecule drug conjugate (SMDC) of DUPA and a duocarmycin built on the solid phase.

In a proof-of-concept study, solid phase synthesis allowed the rapid generation of a small molecule drug conjugate in which the glutamate carboxypeptidase II (GCPII) targeting small molecule DUPA was conjugated to the alkylating subunit of the potent cytotoxin duocarmycin SA. The targeted SMDC contained a cathepsin B cleavable linker, which was shown to be active and selective against cathepsin B over-expressing and GCPII-expressing tumour cell lines.

Diprovocims: A New and Exceptionally Potent Class of Toll-like Receptor Agonists.

A screen conducted with nearly 100000 compounds and a surrogate functional assay for stimulation of an immune response that measured the release of TNF-α from treated human THP-1 myeloid cells differentiated along the macrophage line led to the discovery of the diprovocims. Unique to these efforts and of special interest, the screening leads for this new class of activators of an immune response came from a compound library designed to promote cell-surface receptor dimerization. Subsequent comprehensive structure-activity relationship studies improved the potency 800-fold over that of the screening leads, providing diprovocim-1 and diprovocim-2. The diprovocims act by inducing cell-surface toll-like receptor (TLR)-2 dimerization and activation with TLR1 (TLR1/TLR2 agonist), bear no structural similarity to any known natural or synthetic TLR agonist, and are easy to prepare and synthetically modify, and selected members are active in both human and murine systems. The most potent diprovocim (3, diprovocim-1) elicits full agonist activity at extraordinarily low concentrations (EC50 = 110 pM) in human THP-1 cells, being more potent than the naturally derived TLR1/TLR2 agonist Pam3CSK4 or any other known small molecule TLR agonist.

Adjuvant effect of the novel TLR1/TLR2 agonist Diprovocim synergizes with anti-PD-L1 to eliminate melanoma in mice.

Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti-PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti-PD-L1 treatment.

Duocarmycin SA, a potent antitumor antibiotic, sensitizes glioblastoma cells to proton radiation.

New treatment modalities for glioblastoma multiforme (GBM) are urgently needed. Proton therapy is considered one of the most effective forms of radiation therapy for GBM. DNA alkylating agents such as temozolomide (TMZ) are known to increase the radiosensitivity of GBM to photon radiation. TMZ is a fairly impotent agent, while duocarmycin SA (DSA) is an extremely potent cytotoxic agent capable of inducing a sequence-selective alkylation of duplex DNA. Here, the effects of sub-nM concentrations of DSA on the radiosensitivity of a human GBM cell line (U-138) to proton irradiation were examined. Radiation sensitivity was determined by viability, apoptosis, necrosis and clonogenic assays. DSA concentrations as low as 0.001 nM significantly sensitized U-138 cells to proton irradiation. DSA demonstrates synergistic cytotoxicity against GBM cells treated with proton radiation in vitro, which may represent a novel therapeutic alternative for the treatment of GBM.

High expression of class III ß-tubulin has no impact on functional cancer cell growth inhibition of a series of key vinblastine analogs.

Clinical association studies have implicated high expression of class III ß-tubulin as a predictive factor for lower response rates and reduced overall survival in patients receiving tubulin binding drugs, most notably the taxanes. Because of the implications, we examined a series of key vinblastine analogs that emerged from our studies in functional cell growth inhibition assays for their sensitivity to high expression of class III ß-tubulin (human non-small cell lung cancer cell line A549 vs taxol-resistant A549-T24). Unlike taxol, vinblastine and a set of key analogs 3-10 did not exhibit any loss in sensitivity toward A549-T24. The results suggest that vinblastine and related analogs are not likely prone to resistance derived from high expression of class III ß-tubulin unlike the taxanes. Most significant are the results with 4-6, a subset of 20' amide vinblastine analogs. They match or exceed the potency of vinblastine and they display more potent activity against taxol-resistant A549-T24 than even wild type A549 cells (1.2-2-fold), complementing our prior observations that they also display no sensitivity to overexpression of Pgp (HCT116/VM46 vs HCT116) and are not subject to resistance derived from Pgp efflux.

Vinblastine 20' Amides: Synthetic Analogues That Maintain or Improve Potency and Simultaneously Overcome Pgp-Derived Efflux and Resistance.

A series of 180 vinblastine 20' amides were prepared in three steps from commercially available starting materials, systematically exploring a typically inaccessible site in the molecule enlisting a powerful functionalization strategy. Clear structure-activity relationships and a structural model were developed in the studies which provided many such 20' amides that exhibit substantial and some even remarkable enhancements in potency, many that exhibit further improvements in activity against a Pgp overexpressing resistant cancer cell line, and an important subset of the vinblastine analogues that display little or no differential in activity against a matched pair of vinblastine sensitive and resistant (Pgp overexpressing) cell lines. The improvements in potency directly correlated with target tubulin binding affinity, and the reduction in differential functional activity against the sensitive and Pgp overexpressing resistant cell lines was found to correlate directly with an impact on Pgp-derived efflux.

Key analogs of a uniquely potent synthetic vinblastine that contain modifications of the C20' ethyl substituent.

A key series of vinblastine analogs 7-13, which contain modifications to the C20' ethyl group, was prepared with use of two distinct synthetic approaches that provide modifications of the C20' side chain containing linear and cyclized alkyl groups or added functionalized substituents. Their examination revealed the unique nature of the improved properties of the synthetic vinblastine 6, offers insights into the origins of its increased tubulin binding affinity and 10-fold improved cell growth inhibition potency, and served to probe a small hydrophobic pocket anchoring the binding of vinblastine with tubulin. Especially noteworthy were the trends observed with substitution of the terminal carbon of the ethyl group that, with the exception of 9 (R=F vs H, equipotent), led to remarkably substantial reductions in activity (>10-fold): R=F (equipotent with H)>N3, CN (10-fold)>Me (50-fold)>Et (100-fold)>OH (inactive). This is in sharp contrast to the maintained (7) or enhanced activity (6) observed with its incorporation into a cyclic C20'/C15'-fused six-membered ring.

Design of Benzoxathiazin-3-one 1,1-Dioxides as a New Class of Irreversible Serine Hydrolase Inhibitors: Discovery of a Uniquely Selective PNPLA4 Inhibitor.

The design and examination of 4,1,2-benzoxathiazin-3-one 1,1-dioxides as candidate serine hydrolase inhibitors are disclosed, and represent the synthesis and study of a previously unexplored heterocycle. This new class of activated cyclic carbamates provided selective irreversible inhibition of a small subset of serine hydrolases without release of a leaving group, does not covalently modify active site catalytic cysteine and lysine residues of other enzyme classes, and was found to be amenable to predictable structural modifications that modulate intrinsic reactivity or active site recognition. Even more remarkable and within the small pilot series of candidate inhibitors examined in an initial study, an exquisitely selective inhibitor for a poorly characterized serine hydrolase (PNPLA4, patatin-like phospholipase domain-containing protein 4) involved in adipocyte triglyceride homeostasis was discovered.

Ultrapotent vinblastines in which added molecular complexity further disrupts the target tubulin dimer-dimer interface.

Approaches to improving the biological properties of natural products typically strive to modify their structures to identify the essential pharmacophore, or make functional group changes to improve biological target affinity or functional activity, change physical properties, enhance stability, or introduce conformational constraints. Aside from accessible semisynthetic modifications of existing functional groups, rarely does one consider using chemical synthesis to add molecular complexity to the natural product. In part, this may be attributed to the added challenge intrinsic in the synthesis of an even more complex compound. Herein, we report synthetically derived, structurally more complex vinblastines inaccessible from the natural product itself that are a stunning 100-fold more active (IC50 values, 50-75 pM vs. 7 nM; HCT116), and that are now accessible because of advances in the total synthesis of the natural product. The newly discovered ultrapotent vinblastines, which may look highly unusual upon first inspection, bind tubulin with much higher affinity and likely further disrupt the tubulin head-to-tail α/ß dimer-dimer interaction by virtue of the strategic placement of an added conformationally well-defined, rigid, and extended C20' urea along the adjacent continuing protein-protein interface. In this case, the added molecular complexity was used to markedly enhance target binding and functional biological activity (100-fold), and likely represents a general approach to improving the properties of other natural products targeting a protein-protein interaction.

Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein.

UNLABELLED: Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever. IMPORTANCE: Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.

Synthesis and evaluation of duocarmycin SA analogs incorporating the methyl 1,2,8,8a-tetrahydrocyclopropa[c]imidazolo[4,5-e]indol-4-one-6-carboxylate (CImI) alkylation subunit.

The design, synthesis, and evaluation of methyl 1,2,8,8a-tetrahydrocyclopropa[c]imidazolo[4,5-e]indol-4-one-6-carboxylate (CImI) derivatives are detailed representing analogs of duocarmycin SA and yatakemycin containing an imidazole replacement for the fused pyrrole found in the DNA alkylation subunit.

Total synthesis of vinblastine, related natural products, and key analogues and development of inspired methodology suitable for the systematic study of their structure-function properties.

Biologically active natural products composed of fascinatingly complex structures are often regarded as not amenable to traditional systematic structure-function studies enlisted in medicinal chemistry for the optimization of their properties beyond what might be accomplished by semisynthetic modification. Herein, we summarize our recent studies on the Vinca alkaloids vinblastine and vincristine, often considered as prototypical members of such natural products, that not only inspired the development of powerful new synthetic methodology designed to expedite their total synthesis but have subsequently led to the discovery of several distinct classes of new, more potent, and previously inaccessible analogues. With use of the newly developed methodology and in addition to ongoing efforts to systematically define the importance of each embedded structural feature of vinblastine, two classes of analogues already have been discovered that enhance the potency of the natural products >10-fold. In one instance, remarkable progress has also been made on the refractory problem of reducing Pgp transport responsible for clinical resistance with a series of derivatives made accessible only using the newly developed synthetic methodology. Unlike the removal of vinblastine structural features or substituents, which typically has a detrimental impact, the additions of new structural features have been found that can enhance target tubulin binding affinity and functional activity while simultaneously disrupting Pgp binding, transport, and functional resistance. Already analogues are in hand that are deserving of full preclinical development, and it is a tribute to the advances in organic synthesis that they are readily accessible even on a natural product of a complexity once thought refractory to such an approach.

Inhibition of adenovirus replication by a trisubstituted piperazin-2-one derivative.

The number of disseminated adenovirus (Ad) infections continues to increase mostly due to the growing use of immunosuppressive treatments. Recipients of solid organ or hematopoietic stem cell transplants, mainly in pediatric units, exhibit a high morbidity and mortality due to these infections. Unfortunately, there are no Ad-specific antiviral drugs currently approved for medical use. To address this situation, we used high-throughput screening (HTS) of synthetic small molecule libraries to identify compounds that restrict Ad infection. Among the more than 25,000 compounds screened, we identified a hit compound that significantly inhibited Ad infection. The compound (15D8) is a trisubstituted piperazin-2-one derivative that showed substantial antiviral activity with little or no cytotoxicity at low micromolar concentrations. Compound 15D8 selectively inhibits Ad DNA replication in the nucleus, providing a potential candidate for the development of a new class of antiviral compounds to treat Ad infections.