Hydrophilic Small Molecules That Harness Transthyretin To Enhance the Safety and Efficacy of Targeted Chemotherapeutic Agents.

The hydrophobicity of many chemotherapeutic agents usually results in their nonselective passive distribution into healthy cells and organs causing collateral toxicity. Ligand-targeted drugs (LTDs) are a promising class of targeted anticancer agents. The hydrophilicity of the targeting ligands in LTDs limits its nonselective passive tissue distribution and toxicity to healthy cells. In addition, the small size of LTDs allows for better tumor penetration, especially in the case of solid tumors. However, the short circulation half-life of LTDs, due to their hydrophilicity and small size, remains a significant challenge for achieving their full therapeutic potential. Therefore, extending the circulation half-life of targeted chemotherapeutic agents while maintaining their hydrophilicity and small size will represent a significant advance toward effective and safe cancer treatment. Here, we present a new approach for enhancing the safety and efficacy of targeted chemotherapeutic agents. By endowing hydrophobic chemotherapeutic agents with a targeting moiety and a hydrophilic small molecule that binds reversibly to the serum protein transthyretin, we generated small hydrophilic drug conjugates that displayed enhanced circulation half-life in rodents and selectivity to cancer cells. To the best of our knowledge, this is the first demonstration of a successful approach that maintains the small size and hydrophilicity of targeted anticancer agents containing hydrophobic payloads while at the same time extending their circulation half-life. This was demonstrated by the superior in vivo efficacy and lower toxicity of our conjugates in xenograft mouse models of metastatic prostate cancer.

A biomimetic approach for enhancing the in vivo half-life of peptides.

The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of peptides without compromising their potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo. We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

AG10 inhibits amyloidogenesis and cellular toxicity of the familial amyloid cardiomyopathy-associated V122I transthyretin.

The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Ig light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African American subjects, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 y causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the development of AG10, a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10, combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy.

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine.

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

Peripherally restricted transthyretin-based delivery system for probes and therapeutics avoiding opioid-related side effects.

Several investigations into the sites of action of opioid analgesics have utilized peripherally acting mu-opioid receptor antagonists (PAMORAs), which have been incorrectly assumed to possess limited permeability across the blood-brain barrier. Unfortunately, the poor pharmacokinetic properties of current PAMORAs have resulted in misunderstandings of the role of central nervous system and gastrointestinal tract in precipitating side effects such as opioid-induced constipation. Here, we develop a drug delivery approach for restricting the passage of small molecules across the blood-brain barrier. This allows us to develop naloxone- and oxycodone-based conjugates that display superior potency, peripheral selectivity, pharmacokinetics, and efficacy in rats compared to other clinically used PAMORAs. These probes allow us to demonstrate that the mu-opioid receptors in the central nervous system have a fundamental role in precipitating opioid-induced constipation. Therefore, our conjugates have immediate use as pharmacological probes and potential therapeutic agents for treating constipation and other opioid-related side effects.

Enhancing the Safety and Efficacy of PSMA-Based Small-Molecule Drug Conjugates by Linker Stabilization and Conjugation to Transthyretin Binding Ligand.

This work describes the enhancement of a novel antitumor therapeutic platform that combines advantages from small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). Valine-citrulline (VCit) dipeptide linkers are commonly used cathepsin B cleavable linkers for ADCs. However, the instability of these linkers in mouse serum makes translating efficacy data from mouse to human more challenging. Replacing the VCit linker with glutamic acid-valine-citrulline (EVCit) has been reported to enhance the stability of ADCs in mouse serum. However, the effect of EVCit linker on the stability of SMDCs has not been reported. Here, we report that incorporating the EVCit linker in prostate-specific membrane antigen-targeting SMDCs, equipped with the transthyretin ligand AG10, resulted in conjugates with lower toxicity, an extended half-life, and superior therapeutic efficacy to docetaxel in a xenograft mouse model of prostate cancer. This should make SMDCs' preclinical toxicity and efficacy data from mice more reliable for predicting human results.

Enhancing the Pharmacokinetic Profile of Interleukin 2 through Site-Specific Conjugation to a Selective Small-Molecule Transthyretin Ligand.

Protein drugs hold great promise as therapeutics for a wide range of diseases. Unfortunately, one of the greatest challenges to be addressed during clinical development of protein therapeutics is their short circulation half-life. Several protein conjugation strategies have been developed for half-life extension. However, these strategies have limitations and there remains room for improvement. Here, we report a novel nature-inspired strategy for enhancing the in vivo half-life of proteins. Our strategy involves conjugating proteins to a hydrophilic small molecule that binds reversibly to the plasma protein, transthyretin. We show here that our strategy is effective in enhancing the pharmacokinetic and pharmacodynamic properties of human interleukin 2 in rats, potentially opening the door for more effective and safer cancer immunotherapies. To our knowledge, this is the first example of successful use of a small-molecule that not only extends the half-life but also maintains the smaller size, binding potency, and hydrophilicity of proteins.

Separate entrance and exit portals for ligand traffic in Mycobacterium tuberculosis FabH.

Mycobacterium tuberculosis FabH initiates type II fatty acid synthase-catalyzed formation of the long chain (C(16)-C(22)) acyl-coenzyme A (CoA) precursors of mycolic acids, which are major constituents of the bacterial cell envelope. Crystal structures of M. tuberculosis FabH (mtFabH) show the substrate binding site to be a buried, extended L-shaped channel with only a single solvent access portal. Entrance of an acyl-CoA substrate through the solvent portal would require energetically unfavorable reptational threading of the substrate to its reactive position. Using a class of FabH inhibitors, we have tested an alternative hypothesis that FabH exists in an "open" form during substrate binding and product release, and a "closed" form in which catalysis and intermediate steps occur. This hypothesis is supported by mass spectrometric analysis of the product profile and crystal structures of complexes of mtFabH with these inhibitors.

Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis.

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.

cis-Delta(2,3)-double bond of phoslactomycins is generated by a post-PKS tailoring enzyme.

The antifungal phoslactomycins (PLM A-F), produced by Streptomyces sp. HK803, are structurally unusual in that three of their four double bonds are in the cis form (Delta12,13, Delta14,15, Delta2,3). The PLM polyketide synthase (PKS) has the predicted dehydratase catalytic domain in modules 1, 2, and 5 required for establishing two of these cis double bonds (Delta12,13, Delta14,15), as well as the only trans Delta6,7 double bond. By contrast, the formation of the cis Delta2,3 in the unsaturated lactone moiety of PLMs has presented an enigma because the predicted dehydratase domain in module 7 is absent. Herein, we have demonstrated that the plmT2 gene product, with no homology to PKS dehydratase domains, is required for efficient formation of the cis Delta2,3 alkene. A series of new PLM products in which the C3 hydroxyl group is retained are made in plmT2 deletion mutants. In all of these cases, however, the hydroxyl group is esterified with malonic acid. These malonylated PLM products are converted to the corresponding cis Delta2,3 PLM products and acetic acid by a facile base-catalyzed decarboxylative elimination reaction. Complete or partial restoration of natural PLM production in a plmT2 deletion mutant can be accomplished by plasmid based expression of plmT2 or fos ORF4 (a homologous gene from the fostriecin biosynthetic gene cluster), respectively. The data indicate that dehydratase-independent pathways also function in establishment of unsaturated 6-membered lactone moieties in other PKS pathways and provide the first biosynthetic insights into the possible routes by which unusual malonylated polyketide products are generated.

Alkyl-CoA disulfides as inhibitors and mechanistic probes for FabH enzymes.

The first step of the reaction catalyzed by the homodimeric FabH from a dissociated fatty acid synthase is acyl transfer from acyl-CoA to an active site cysteine. We report that C1 to C10 alkyl-CoA disulfides irreversibly inhibit Escherichia coli FabH (ecFabH) and Mycobacterium tuberculosis FabH with relative efficiencies that reflect these enzymes' differential acyl-group specificity. Crystallographic and kinetic studies with MeSSCoA show rapid inhibition of one monomer of ecFabH through formation of a methyl disulfide conjugate with this cysteine. Reaction of the second subunit with either MeSSCoA or acetyl-CoA is much slower. In the presence of malonyl-ACP, the acylation rate of the second subunit is restored to that of the native ecFabH. These observations suggest a catalytic model in which a structurally disordered apo-ecFabH dimer orders on binding either the first substrate, acetyl-CoA, or the inhibitor MeSSCoA, and is restored to a disordered state on binding of malonyl-ACP.

Synthesis and biological evaluation of novel sulfonyl-naphthalene-1,4-diols as FabH inhibitors.

A series of analogs of 2-tosylnaphthalene-1,4-diol were prepared and were found to be potent 10-20 nM reversible inhibitors of the Escherichia coli FabH enzyme. The inhibitors were also effective but to a lesser degree (30 nM-5 microM), against the Mycobacterium tuberculosis and Plasmodium falciparum FabH enzymes. Preliminary SAR studies demonstrated that the sulfonyl group and naphthalene-1,4 diol were required for activity against all enzymes but the toluene portion could be significantly altered and leads to either modest increases or decreases in activity against the three enzymes. The in vitro activity of the analogs against E. coli FabH parallel the in vivo activity against E. coli TolC strain and many of the compounds were also shown to have antimalarial activity against P. falciparum.

Unsymmetric aryl-alkyl disulfide growth inhibitors of methicillin-resistant Staphylococcus aureus and Bacillus anthracis.

This study describes the antibacterial properties of synthetically produced mixed aryl-alkyl disulfide compounds as a means to control the growth of Staphylococcus aureus and Bacillus anthracis. Some of these compounds exerted strong in vitro bioactivity. Our results indicate that among the 12 different aryl substituents examined, nitrophenyl derivatives provide the strongest antibiotic activities. This may be the result of electronic activation of the arylthio moiety as a leaving group for nucleophilic attack on the disulfide bond. Small alkyl residues on the other sulfur provide the best activity as well, which for different bacteria appears to be somewhat dependent on the nature of the alkyl moiety. The mechanism of action of these lipophilic disulfides is likely similar to that of previously reported N-thiolated beta-lactams, which have been shown to produce alkyl-CoA disulfides through a thiol-disulfide exchange within the cytoplasm, ultimately inhibiting type II fatty acid synthesis. However, the mixed alkyl-CoA disulfides themselves show no antibacterial activity, presumably due to the inability of the highly polar compounds to cross the bacterial cell membrane. These structurally simple disulfides have been found to inhibit beta-ketoacyl-acyl carrier protein synthase III, or FabH, a key enzyme in type II fatty acid biosynthesis, and thus may serve as new leads to the development of effective antibacterials for MRSA and anthrax infections.

Probing reactivity and substrate specificity of both subunits of the dimeric Mycobacterium tuberculosis FabH using alkyl-CoA disulfide inhibitors and acyl-CoA substrates.

The dimeric Mycobacterium tuberculosis FabH (mtFabH) catalyses a Claisen-type condensation between an acyl-CoA and malonyl-acyl carrier protein (ACP) to initiate the Type II fatty acid synthase cycle. To analyze the initial covalent acylation of mtFabH with acyl-CoA, we challenged it with mixture of C6-C20 acyl-CoAs and the ESI-MS analysis showed reaction at both subunits and a strict specificity for C12 acyl CoA. Crystallographic and ESI-MS studies of mtFabH with a decyl-CoA disulfide inhibitor revealed a decyl chain bound in acyl-binding channels of both subunits through disulfide linkage to the active site cysteine. These data provide the first unequivocal evidence that both subunits of mtFabH can react with substrates or inhibitor. The discrepancy between the observed C12 acyl-CoA substrate specificity in the initial acylation step and the higher catalytic efficiency of mtFabH for C18-C20 acyl-CoA substrates in the overall mtFabH catalyzed reaction suggests a role for M. tuberculosis ACP as a specificity determinant in this reaction.

Design, total synthesis, and evaluation of novel open-chain epothilone analogues.

[structure: see text] The design, total synthesis, and biological evaluation of two open-chain analogues of epothilone incorporating the critical C1-C8 fragment and the aromatic side chain held together by a small molecular scaffold have been achieved. Biological evaluation revealed that further restraint between the flexible C1-C8 region and the molecular scaffold may be necessary for potent inhibition of cell proliferation.

Potent kinetic stabilizers that prevent transthyretin-mediated cardiomyocyte proteotoxicity.

A valine-to-isoleucine mutation at position 122 of the serum protein transthyretin (TTR), found in 3 to 4% of African Americans, alters its stability, leading to amyloidogenesis and cardiomyopathy. In addition, 10 to 15% of individuals older than 65 years develop senile systemic amyloidosis and cardiac TTR deposits because of wild-type TTR amyloidogenesis. Although several drugs are in development, no approved therapies for TTR amyloid cardiomyopathy are yet available, so the identification of additional compounds that prevent amyloid-mediated cardiotoxicity is needed. To this aim, we developed a fluorescence polarization-based high-throughput screen and used it to identify several new chemical scaffolds that target TTR. These compounds were potent kinetic stabilizers of TTR and prevented TTR tetramer dissociation, partial unfolding, and aggregation of both wild type and the most common cardiomyopathy-associated TTR mutant, V122I-TTR. High-resolution co-crystal structures and characterization of the binding energetics revealed how these diverse structures bound to tetrameric TTR. These compounds effectively inhibited the proteotoxicity of V122I-TTR toward human cardiomyocytes. Several of these ligands stabilized TTR in human serum more effectively than diflunisal, which is a well-studied inhibitor of TTR aggregation, and may be promising leads for the treatment or prevention of TTR-mediated cardiomyopathy.

Total synthesis and selective activity of a new class of conformationally restrained epothilones.

Stereoselective total syntheses of two novel conformationally restrained epothilone analogues are described. Evans asymmetric alkylation, Brown allylation, and a diastereoselective aldol reaction served as the key steps in the stereoselective synthesis of one of the two key fragments of the convergent synthetic approach. Enzyme resolution was employed to obtain the second fragment as a single enantiomer. The molecules were assembled by esterification, followed by ring-closing metathesis. In preliminary cytotoxicity studies, one of the analogues showed strong and selective growth inhibitory activity against two leukemia cell lines over solid human tumor cell lines. The precise biological mechanism of action and high degree of selectivity of this analogue remain to be examined.

Synthesis and biological evaluation of thiazolidine-2-one 1,1-dioxide as inhibitors of Escherichia coli beta-ketoacyl-ACP-synthase III (FabH).

A series of cyclic sulfones has been synthesized and their activity against beta-ketoacyl-ACP-synthase III (FabH) has been investigated. The compounds are selectively active against Escherichia coli FabH (ecFabH), but not Mycobacterium tuberculosis FabH (mtFabH) or Plasmodium falciparum KASIII (PfKASIII). The activity against ecFabH ranges from 0.9 to >100microM and follows a consistent general SAR trend. Many of the compounds were shown to have antimalarial activity against chloroquine (CQ)-sensitive (D6) P. falciparum (IC(50)=5.3microM for the most potent inhibitor) and some were active against E. coli (MIC=6.6microg/ml for the most potent inhibitor).