Guanine-containing ssDNA and RNA induce dimeric and tetrameric structural forms of SAMHD1.

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish in cryo-EM and biochemical studies that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ∼20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

Guanine-containing ssDNA and RNA induce dimeric and tetrameric SAMHD1 in cryo-EM and binding studies.

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with ~20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding.

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

Macromolecular crowding induces compaction and DNA binding in the disordered N-terminal domain of hUNG2.

Many human DNA repair proteins have disordered domains at their N- or C-termini with poorly defined biological functions. We recently reported that the partially structured N-terminal domain (NTD) of human uracil DNA glycosylase 2 (hUNG2), functions to enhance DNA translocation in crowded environments and also targets the enzyme to single-stranded/double-stranded DNA junctions. To understand the structural basis for these effects we now report high-resolution heteronuclear NMR studies of the isolated NTD in the presence and absence of an inert macromolecular crowding agent (PEG8K). Compared to dilute buffer, we find that crowding reduces the degrees of freedom for the structural ensemble, increases the order of a PCNA binding motif and dramatically promotes binding of the NTD for DNA through a conformational selection mechanism. These findings shed new light on the function of this disordered domain in the context of the crowded nuclear environment.

Rapamycin-inspired macrocycles with new target specificity.

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.

Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action.

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module's molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.

Structure-activity relationship study of itraconazole, a broad-range inhibitor of picornavirus replication that targets oxysterol-binding protein (OSBP).

Itraconazole (ITZ) is a well-known, FDA-approved antifungal drug that is also in clinical trials for its anticancer activity. ITZ exerts its anticancer activity through several disparate targets and pathways. ITZ inhibits angiogenesis by hampering the functioning of the vascular endothelial growth receptor 2 (VEGFR2) and by indirectly inhibiting mTOR signaling. Furthermore, ITZ directly inhibits the growth of several types of tumor cells by antagonizing Hedgehog signaling. Recently, we reported that ITZ also has broad-spectrum antiviral activity against enteroviruses, cardioviruses and hepatitis C virus, independent of established ITZ-activities but instead via a novel target, oxysterol-binding protein (OSBP), a cellular lipid shuttling protein. In this study, we analyzed which structural features of ITZ are important for the OSBP-mediated antiviral activity. The backbone structure, consisting of five rings, and the sec-butyl chain are important for antiviral activity, whereas the triazole moiety, which is critical for antifungal activity, is not. The features required for OSBP-mediated antiviral activity of ITZ overlap mostly with published features required for inhibition of VEGFR2 trafficking, but not Hh signaling. Furthermore, we use in silico studies to explore how ITZ could bind to OSBP. Our data show that several pharmacological activities of ITZ can be uncoupled, which is a critical step in the development of ITZ-based antiviral compounds with greater specificity and reduced off-target effects.

Pyridinylquinazolines selectively inhibit human methionine aminopeptidase-1 in cells.

Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1-4), but all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1-4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.

Tricyclic thiazoles are a new class of angiogenesis inhibitors.

Tricyclic thiazoleamine derivatives that were identified as hits in a screen against human umbilical vein endothelial cell proliferation were subjected to a structure-activity relationship study. Two structurally superimposable scaffolds-4H-thiochromeno[4,3-d]thiazol-2-amine and 5,6-dihydro-4H-benzo[6,7]cyclohepta[1,2-d]thiazol-2-amine derivatives-yielded low-micromolar inhibitors, and two among them 37 and 43 also exhibited antiangiogenic activity in an endothelial tube formation assay. Thus, 37 and 43 can serve as leads to develop a novel class of antiangiogenic agents.

Pyridinylpyrimidines selectively inhibit human methionine aminopeptidase-1.

Cellular protein synthesis is initiated with methionine in eukaryotes with few exceptions. Methionine aminopeptidases (MetAPs) which catalyze the process of N-terminal methionine excision are essential for all organisms. In mammals, type 2 MetAP (MetAP2) is known to be important for angiogenesis, while type 1 MetAP (MetAP1) has been shown to play a pivotal role in cell proliferation. Our previous high-throughput screening of a commercial compound library uncovered a novel class of inhibitors for both human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2). This class of inhibitors contains a pyridinylpyrimidine core. To understand the structure-activity relationship (SAR) and to search for analogues of 2 with greater potency and higher HsMetAP1-selectivity, a total of 58 analogues were acquired through either commercial source or by in-house synthesis and their inhibitory activities against HsMetAP1 and HsMetAP2 were determined. Through this systematic medicinal chemistry analysis, we have identified (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of HsMetAP1; (2) 5'-chloro as the favored substituent on the pyridine ring for the enhanced potency against HsMetAP1; and (3) long C4 side chains as the essentials for higher HsMetAP1-selectivity. At the end of our SAR campaign, 25b, 25c, 26d and 30a-30c are among the most selective and potent inhibitors of purified HsMetAP1 reported to date. In addition, we also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1.

Salicylanilide derivatives block Mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase.

The global burden of tuberculosis, its health and socio-economic impacts, the presence of drug-resistant forms and a potential threat of latent tuberculosis should serve as a strong impetus for the development of novel antituberculosis agents. We reported the in vitro activity of salicylanilide benzoates and pyrazine-2-carboxylates against Mycobacterium tuberculosis (minimum inhibitory concentrations as low as 0.5 µmol/L). Nineteen salicylanilide derivatives with mostly good antimycobacterial activity were evaluated for the inhibition of two essential mycobacterial enzymes, methionine aminopeptidase and isocitrate lyase, which are necessary for the maintenance of the latent tuberculosis infection. Salicylanilide derivatives act as moderate inhibitors of both mycobacterial and human methionine aminopeptidase and they also affect the function of mycobacterial isocitrate lyase. 4-Bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate was the most potent inhibitor of mycobacterial methionine aminopeptidase (41% inhibition at 10 µmol/L) and exhibited the highest selectivity. 5-Chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide and 4-chloro-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl pyrazine-2-carboxylate caused 59% inhibition of isocitrate lyase at 100 µmol/L concentration and (S)-4-bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl 2-acetamido-3-phenylpropanoate produced 22% inhibition at 10 µmol/L; this rate is approximately comparable to 3-nitropropionic acid. Inhibition of those enzymes contributes at least in part to the antimicrobial activity of the compounds.

Analogs of N'-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide inhibit Mycobacterium tuberculosis methionine aminopeptidases.

Our previous target validation studies established that inhibition of methionine aminopeptidases (MtMetAP, type 1a and 1c) from Mycobacterium tuberculosis (Mtb) is an effective approach to suppress Mtb growth in culture. A novel class of MtMetAP1c inhibitors comprising of N'-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide (4c) was uncovered through a high-throughput screen (HTS). A systematic structure-activity relationship study (SAR) yielded variants of the hit, 4b, 4h, and 4k, bearing modified A- and B-rings as potent inhibitors of both MtMetAPs. Except methanimidamide 4h that showed a moderate Mtb inhibition, a desirable minimum inhibitory concentration (MIC) was not obtained with the current set of MtMetAP inhibitors. However, the SAR data generated thus far may prove valuable for further tuning of this class of inhibitors as effective anti-tuberculosis agents.

Substituted oxines inhibit endothelial cell proliferation and angiogenesis.

Two substituted oxines, nitroxoline (5) and 5-chloroquinolin-8-yl phenylcarbamate (22), were identified as hits in a high-throughput screen aimed at finding new anti-angiogenic agents. In a previous study, we have elucidated the molecular mechanism of antiproliferative activity of nitroxoline in endothelial cells, which comprises of a dual inhibition of type 2 human methionine aminopeptidase (MetAP2) and sirtuin 1 (SIRT1). Structure-activity relationship study (SAR) of nitroxoline offered many surprises where minor modifications yielded oxine derivatives with increased potency against human umbilical vein endothelial cells (HUVEC), but with entirely different as yet unknown mechanisms. For example, 5-nitrosoquinolin-8-ol (33) inhibited HUVEC growth with sub-micromolar IC(50), but did not affect MetAP2 or MetAP1, and it only showed weak inhibition against SIRT1. Other sub-micromolar inhibitors were derivatives of 5-aminoquinolin-8-ol (34) and 8-sulfonamidoquinoline (32). A sulfamate derivative of nitroxoline (48) was found to be more potent than nitroxoline with the retention of activities against MetAP2 and SIRT1. The bioactivity of the second hit, micromolar HUVEC and MetAP2 inhibitor carbamate 22 was improved further with an SAR study culminating in carbamate 24 which is a nanomolar inhibitor of HUVEC and MetAP2.

Novel structurally related compounds reactivate latent HIV-1 in a bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation.

BACKGROUND: The latent reservoir of HIV-1 in resting memory CD4+ T cells is a major barrier to curing HIV-1 infection. Eradication strategies involve reactivation of this latent reservoir; however, agents that reactivate latent HIV-1 through non-specific T cell activation are toxic. METHODS: Using latently infected Bcl-2-transduced primary CD4+ T cells, we screened the MicroSource Spectrum library for compounds that reactivate latent HIV-1 without global T cell activation. Based on the structures of the initial hits, we assembled ∼50 derivatives from commercial sources and mostly by synthesis. The dose-response relationships of these derivatives were established in a primary cell model. Activities were confirmed with another model of latency (J-Lat). Cellular toxicity and cytokine secretion were tested using freshly isolated human CD4+ T cells. RESULTS: We identified two classes of quinolines that reactivate latent HIV-1. Class I compounds are the Mannich adducts of 5-chloroquinolin-8-ol. Class II compounds are quinolin-8-yl carbamates. Most EC(50) values were in the 0.5-10 µM range. HIV-1 reactivation ranged from 25% to 70% for anti-CD3+ anti-CD28 co-stimulation. All quinolin-8-ol derivatives that reactivate latent HIV-1 follow Lipinski's Rule of Five, and most follow the stricter rule of three for leads. After 48 h of treatment, none of the analogues induced detectable cytokine secretion in primary resting CD4+ T cells. CONCLUSIONS: We discovered a group of quinolin-8-ol derivatives that can induce latent HIV-1 in a primary cell model without causing global T cell activation. This work expands the number of latency-reversing agents and provides new possible scaffolds for further drug development research.

Optimization of the potency and pharmacokinetic properties of a macrocyclic ghrelin receptor agonist (Part I): Development of ulimorelin (TZP-101) from hit to clinic.

High-throughput screening of Tranzyme Pharma's proprietary macrocycle library using the aequorin Ca2+-bioluminescence assay against the human ghrelin receptor (GRLN) led to the discovery of novel agonists against this G-protein coupled receptor. Early hits such as 1 (Ki=86 nM, EC50=134 nM) though potent in vitro displayed poor pharmacokinetic properties that required optimization. While such macrocycles are not fully rule-of-five compliant, principally due to their molecular weight and clogP, optimization of their pharmacokinetic properties proved feasible largely through conformational rigidification. Extensive SAR led to the identification of 2 (Ki=16 nM, EC50=29 nM), also known as ulimorelin or TZP-101, which has progressed to phase III human clinical trials for the treatment of postoperative ileus. X-ray structure and detailed NMR studies indicated a rigid peptidomimetic portion in 2 that is best defined as a nonideal type-I' ß-turn. Compound 2 is 24% orally bioavailable in both rats and monkeys. Despite its potency, in vitro and in gastric emptying studies, 2 did not induce growth hormone (GH) release in rats, thus demarcating the GH versus GI pharmacology of GRLN.

Characterization of clioquinol and analogues as novel inhibitors of methionine aminopeptidases from Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis claims about five thousand lives daily world-wide, while one-third of the world is infected with dormant tuberculosis. The increased emergence of multi- and extensively drug-resistant strains of M. tuberculosis (Mtb) has heightened the need for novel antimycobacterial agents. Here, we report the discovery of 7-bromo-5-chloroquinolin-8-ol (CLBQ14)-a congener of clioquinol (CQ) as a potent and selective inhibitor of two methionine aminopeptidases (MetAP) from M. tuberculosis: MtMetAP1a and MtMetAP1c. MetAP is a metalloprotease that removes the N-terminal methionine during protein synthesis. N-terminal methionine excision (NME) is a universally conserved process required for the post-translational modification of a significant part of the proteome. The essential role of MetAP in microbes makes it a promising target for the development of new therapeutics. Using a target-based approach in a high-throughput screen, we identified CLBQ14 as a novel MtMetAP inhibitor with higher specificity for both MtMetAP1s relative to their human counterparts. We also found that CLBQ14 is potent against replicating and aged non-growing Mtb at low micro molar concentrations. Furthermore, we observed that the antimycobacterial activity of this pharmacophore correlates well with in vitro enzymatic inhibitory activity. Together, these results revealed a new mode of action of clioquinol and its congeners and validated the therapeutic potential of this pharmacophore for TB chemotherapy.

A simple and efficient route to the FKBP-binding domain from rapamycin.

A simple and highly efficient route to the FKBP-binding domain (FKBD) from the natural product rapamycin has been developed, which entails a sequence of ozonolysis/Baeyer-Villiger/Wittig reactions. The newly synthesized FKBD may serve as a core to assemble hybrid macrocyclic libraries for the discovery of novel probes of protein function and to synthesize new ligands for the FKBP family of proteins.

Diazo reagents with small steric footprints for simultaneous arming/SAR studies of alcohol-containing natural products via O-H insertion.

Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely, an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O-H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated, and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of α-trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors.

Inhibition of eukaryotic translation elongation by the antitumor natural product Mycalamide B.

Mycalamide B (MycB) is a marine sponge-derived natural product with potent antitumor activity. Although it has been shown to inhibit protein synthesis, the molecular mechanism of action by MycB remains incompletely understood. We verified the inhibition of translation elongation by in vitro HCV IRES dual luciferase assays, ribosome assembly, and in vivo [(35)S]methinione labeling experiments. Similar to cycloheximide (CHX), MycB inhibits translation elongation through blockade of eEF2-mediated translocation without affecting the eEF1A-mediated loading of tRNA onto the ribosome, AUG recognition, or dipeptide synthesis. Using chemical footprinting, we identified the MycB binding site proximal to the C3993 28S rRNA residue on the large ribosomal subunit. However, there are also subtle, but significant differences in the detailed mechanisms of action of MycB and CHX. First, MycB arrests the ribosome on the mRNA one codon ahead of CHX. Second, MycB specifically blocked tRNA binding to the E-site of the large ribosomal subunit. Moreover, they display different polysome profiles in vivo. Together, these observations shed new light on the mechanism of inhibition of translation elongation by MycB.

Disulfiram reactivates latent HIV-1 in a Bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation.

Highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 levels to below the detection limit. However, due to the latent reservoir in resting CD4(+) cells, HAART is not curative. Elimination of this reservoir is critical to curing HIV-1 infection. Agents that reactivate latent HIV-1 through nonspecific T cell activation are toxic. Here we demonstrate in a primary CD4(+) T cell model that the FDA-approved drug disulfiram reactivates latent HIV-1 without global T cell activation. The extent to which disulfiram reactivates latent HIV-1 in patient cells is unclear, but the drug alone or in combination may be useful in future eradication strategies.