We recently reported on a controlled deactivation/detoxification approach for obtaining cannabinoids with improved druggability. Our design incorporates a metabolically labile ester group at strategic positions within the THC structure. We have now synthesized a series of (−)-Δ8-THC analogues encompassing a carboxyester group within the 3-alkyl chain in an effort to explore this novel cannabinergic chemo-type for CB receptor binding affinity, in vitro and in vivo potency and efficacy, as well as controlled deactivation by plasma esterases. We have also probed the chain’s polar characteristics with regard to fast onset and short duration of action. Our lead molecule, namely 2-[(6aR,10aR)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-3-yl]-2-methyl-propanoic acid 3-cyano-propyl ester (AM7438), showed picomolar affinity for CB receptors and is deactivated by plasma esterases while the respective acid metabolite is inactive. In further in vitro and in vivo experiments, the compound was found to be a remarkably potent and efficacious CB1 receptor agonist with relatively fast onset/offset of action.
Previously, we reported that Akt inactivation by γ-tocopherol (2) in PTEN-negative prostate cancer cells resulted from its unique ability to facilitate membrane co-localization of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase isoform 1), a Ser473-specific Akt phosphatase, through pleckstrin homology (PH) domain binding. This finding provided a basis for exploiting 2 to develop a novel class of PHLPP1-targeted Akt inhibitors. Here, we used 3 (γ-VE5), a side chain-truncated 2 derivative, as a scaffold for lead optimization. The proof-of-concept of this structural optimization was obtained by 20, which exhibited higher antitumor efficacy than 3 in PTEN-negative cancer cells through PHLPP1-facilitated Akt inactivation. Like 3, 20 preferentially recognized the PH domains of Akt and PHLPP1, as its binding affinities for other PH domains, including those of ILK and PDK1, were an order-of-magnitude lower. Moreover, 20 was orally active in suppressing xenograft tumor growth in nude mice, which underlines the translational potential of this new class of Akt inhibitor in PTEN-deficient cancers.
Undesirable side effects associated
with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R),
a tractable target for treating several pathologies affecting humans,
have greatly limited their translational potential. Recent discovery
of CB1R negative allosteric modulators (NAMs) has renewed interest
in CB1R by offering a potentially safer therapeutic avenue. To elucidate
the CB1R allosteric binding motif and thereby facilitate rational
drug discovery, we report the synthesis and biochemical characterization
of first covalent ligands designed to bind irreversibly to the CB1R
allosteric site. Either an electrophilic or a photoactivatable group
was introduced at key positions of two classical CB1R NAMs: Org27569
(1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays,
did not exhibit inverse agonism, and behaved as a robust positive
allosteric modulator of binding of orthosteric agonist CP55,940. This
novel covalent probe can serve as a useful tool for characterizing
CB1R allosteric ligand-binding motifs.
2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]-pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.
A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6–11 with varying chain lengths (n = 1–6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6–11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8–10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure–activity relationship for transport and dual target inhibition.
Linear 2-alkylaminoethyl-1,1-bisphosphonates are effective agents against proliferation of Trypanosoma cruzi--the etiologic agent of American trypanosomiasis (Chagas disease)--exhibiting IC50 values in the nanomolar range against the parasites. This activity is associated with inhibition at the low nanomolar level of the T. cruzi farnesyl diphosphate synthase (TcFPPS). X-ray structures and thermodynamic data of the complexes TcFPPS with five compounds of this family show that the inhibitors bind to the allylic site of the enzyme with their alkyl chain occupying the cavity that binds the isoprenoid chain of the substrate. The compounds bind to TcFPPS with unfavorable enthalpy compensated by a favorable entropy that results from a delicate balance between two opposing effects: the loss of conformational entropy due to freezing of single bond rotations, and the favorable burial of the hydrophobic alkyl chains. The data suggest that introduction of strategically placed double bonds and methyl branches should increase affinity substantially.
Bisphosphonate; Trypanosoma cruzi; Chagas disease; farnesyl diphosphate synthase; FPPS; IPP; DMAPP; ITC; farnesyl pyrophosphate synthase; mevalonate pathway
Structural coverage of the human kinome has been steadily increasing over time. The structures provide valuable insights into the molecular basis of kinase function and also provide a foundation for understanding the mechanisms of kinase inhibitors. There are a large number of kinase structures in the PDB for which the Asp and Phe of the DFG motif on the activation loop swap positions, resulting in the formation of a new allosteric pocket. We refer to these structures as “classical DFG-out” conformations in order to distinguish them from conformations which have also been referred to as DFG-out in the literature but which do not have a fully formed allosteric pocket. We have completed a structural analysis of almost two hundred small molecule inhibitors bound to classical DFG-out conformations; we find that they are recognized by both type I and type II inhibitors. In contrast, we find that non-classical DFG-out conformations strongly select against type II inhibitors because these structures have not formed a large enough allosteric pocket to accommodate this type of binding mode. In the course of this study we discovered that the number of structurally validated type II inhibitors that can be found in the PDB and that are also represented in publicly available biochemical profiling studies of kinase inhibitors is very small. We have obtained new profiling results for several additional structurally validated type II inhibitors identified through our conformational analysis. Although the available profiling data for type II inhibitors is still much smaller than for type I inhibitors, a comparison of the two datasets supports the conclusion that type II inhibitors are more selective than type I. We comment on the possible contribution of the DFG-in to DFG-out conformational reorganization to the selectivity.
Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential or receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.
receptor β (ERβ) selective agonists are considered potential
therapeutic agents for a variety of pathological conditions, including
several types of cancer. Their development is particularly challenging,
since differences in the ligand binding cavities of the two ER subtypes
α and β are minimal. We have carried out a rational design
of new salicylketoxime derivatives which display unprecedentedly high
levels of ERβ selectivity for this class of compounds, both
in binding affinity and in cell-based functional assays. An endogenous
gene expression assay was used to further characterize the pharmacological
action of these compounds. Finally, these ERβ-selective agonists
were found to inhibit proliferation of a glioma cell line in vitro.
Most importantly, one of these compounds also proved to be active
in an in vivo xenograft model of human glioma, thus demonstrating
the high potential of this type of compounds against this devastating
arginine deiminases (PADs) catalyze the post-translational
hydrolysis of arginine residues to form citrulline. This once obscure
modification is now known to play a key role in the etiology of multiple
autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis,
lupus, and ulcerative colitis) and in some forms of cancer. Among
the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which
isozyme contributes to disease pathogenesis. Toward the identification
of potent, selective, and bioavailable PAD inhibitors that can be
used to elucidate the specific roles of each isozyme, we describe
tetrazole analogs as suitable backbone amide bond bioisosteres for
the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs
are highly potent and show selectivity toward particular isozymes.
Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell
killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell
line and can also block the formation of neutrophil extracellular
traps. These bioisosteres represent an important step in our efforts
to develop stable, bioavailable, and selective inhibitors for the
Protein arginine methyltransferase
1 (PRMT1) is involved in many biological activities, such as gene
transcription, signal transduction, and RNA processing. Overexpression
of PRMT1 is related to cardiovascular diseases, kidney diseases, and
cancers; therefore, selective PRMT1 inhibitors serve as chemical probes
to investigate the biological function of PRMT1 and drug candidates
for disease treatment. Our previous work found trimethine cyanine
compounds that effectively inhibit PRMT1 activity. In our present
study, we systematically investigated the structure–activity
relationship of cyanine structures. A pentamethine compound, E-84
(compound 50), showed inhibition on PRMT1 at the micromolar
level and 6- to 25-fold selectivity over CARM1, PRMT5, and PRMT8.
The cellular activity suggests that compound 50 permeated
the cellular membrane, inhibited cellular PRMT1 activity, and blocked
leukemia cell proliferation. Additionally, our molecular docking study
suggested compound 50 might act by occupying the cofactor
binding site, which provided a roadmap to guide further optimization
of this lead compound.
Four side chain fluorinated analogues of 1α,25-dihydroxy-19-norvitamin D have been prepared in convergent syntheses using the Wittig-Horner reaction as a key step. Structures and absolute configurations of analogues 3 and 5 were confirmed by X-ray crystallography. All analogues showed high potency in HL-60 cell differentiation and vitamin D-24-hydroxylase (24-OHase) transcription as compared to 1α,25-dihydroxyvitamin D3 (1). Most important is that all of the 20S-configured derivatives (4 and 6) had high bone mobilizing activity in vivo. However, in the 20R series, a 2-methylene group was required for high bone mobilizing activity. A change in positioning of the 20R molecule in the vitamin D receptor when the 2-methylene group is present may provide new insight into the molecular basis of bone calcium mobilization induced by vitamin D.
Vitamin D analogues; 19-Norvitamin D; Vitamin D receptor; Cellular HL-60 differentiation; calcemic activity; CYP24A1 hydroxylase; 24-OHase transcription
A series of fluorine-containing PDE10A inhibitors were designed and synthesized to improve the metabolic stability of [11C]MP-10. Twenty of the 22 new analogues had high potency and selectivity for PDE10A: 18a–j, 19d–j, 20a–b, and 21b had IC50 values <5 nM for PDE10A. Seven F-18 labeled compounds [18F]18a–e, [18F]18g, and [18F]20a were radiosynthesized by 18F-introduction onto the quinoline rather than the pyrazole moiety of the MP-10 pharmacophore and performed in vivo evaluation. Biodistribution studies in rats showed ~2-fold higher activity in the PDE10A-enriched striatum than nontarget brain regions; this ratio increased from 5 to 30 min postinjection, particularly for [18F]18a–d and [18F]20a. Micro-PET studies of [18F]18d and [18F]20a in nonhuman primates provided clear visualization of striatum with suitable equilibrium kinetics and favorable metabolic stability. These results suggest this strategy may identify a 18F-labeled PET tracer for quantifying the levels of PDE10A in patients with CNS disorders including Huntington’s disease and schizophrenia.
A quinazolinedione-derived screening hit 2 was discovered with cellular antiviral activity against respiratory syncytial virus (CPE EC50 = 2.1 µM), moderate efficacy in reducing viral progeny (4.2 log at 10 µM), and marginal cytotoxic liability (selectivity index, SI ~ 24). Scaffold optimization delivered analogs with improved potency and selectivity profiles. Most notable were compounds 15 and 19 (EC50 = 300–500 nM, CC50 > 50 µM, SI > 100), which significantly reduced viral titer (>400,000-fold), and several analogs were shown to block the activity of the RNA-dependent RNA-polymerase complex of RSV.
We have previously reported that
stabilization of the G-quadruplex
structures in the HIV-1 long terminal repeat (LTR) promoter suppresses
viral transcription. Here we sought to develop new G-quadruplex ligands
to be exploited as antiviral compounds by enhancing binding toward
the viral G-quadruplex structures. We synthesized naphthalene diimide
derivatives with a lateral expansion of the aromatic core. The new
compounds were able to bind/stabilize the G-quadruplex to a high extent,
and some of them displayed clear-cut selectivity toward the viral
G-quadruplexes with respect to the human telomeric G-quadruplexes.
This feature translated into low nanomolar anti-HIV-1 activity toward
two viral strains and encouraging selectivity indexes. The selectivity
depended on specific recognition of LTR loop residues; the mechanism
of action was ascribed to inhibition of LTR promoter activity in cells.
This is the first example of G-quadruplex ligands that show increased
selectivity toward the viral G-quadruplexes and display remarkable
The PLP-dependent transaminase (BioA) of Mycobacterium
tuberculosis and other pathogens that catalyzes the second step of
biotin biosynthesis is a now well-validated target for antibacterial
development. Fragment screening by differential scanning fluorimetry has been
performed to discover new chemical scaffolds and promote optimization of
existing inhibitors. Calorimetry confirms binding of six molecules with high
ligand efficiency. Thermodynamic data identifies which molecules bind with the
enthalpy driven stabilization preferred in compounds that represent attractive
starting points for future optimization. Crystallographic characterization of
complexes with these molecules reveals the dynamic nature of the BioA active
site. Different side chain conformational states are stabilized in response to
binding by different molecules. A detailed analysis of conformational diversity
in available BioA structures is presented, resulting in the identification of
two states that might be targeted with molecular scaffolds incorporating
well-defined conformational attributes. This new structural data can be used as
part of a scaffold hopping strategy to further optimize existing inhibitors or
create new small molecules with improved therapeutic potential.
Reverse transcriptase (RT) associated
ribonuclease H (RNase H) remains the only virally encoded enzymatic
function not targeted by current chemotherapy against human immunodeficiency
virus (HIV). Although numerous chemotypes have been reported to inhibit
HIV RNase H biochemically, few show significant antiviral activity
against HIV. We report herein the design, synthesis, and biological
evaluations of a novel variant of 2-hydroxyisoquinoline-1,3-dione
(HID) scaffold featuring a crucial C-6 benzyl or biarylmethyl moiety.
The synthesis involved a recently reported metal-free direct benzylation
between tosylhydrazone and boronic acid, which allowed the generation
of structural diversity for the hydrophobic aromatic region. Biochemical
studies showed that the C-6 benzyl and biarylmethyl HID analogues,
previously unknown chemotypes, consistently inhibited HIV RT-associated
RNase H and polymerase with IC50s in low to submicromolar
range. The observed dual inhibitory activity remained uncompromised
against RT mutants resistant to non-nucleoside RT inhibitors (NNRTIs),
suggesting the involvement of binding site(s) other than the NNRTI
binding pocket. Intriguingly, these same compounds inhibited the polymerase,
but not the RNase H function of Moloney Murine Leukemia Virus (MoMLV)
RT and also inhibited Escherichia coli RNase H. Additional biochemical testing revealed a substantially
reduced level of inhibition against HIV integrase. Molecular docking
corroborates favorable binding of these analogues to the active site
of HIV RNase H. Finally, a number of these analogues also demonstrated
antiviral activity at low micromolar concentrations.
of bacterial nitric oxide synthase (bNOS) has the potential to improve
the efficacy of antimicrobials used to treat infections by Gram-positive
pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity
toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge
because of the near identical NOS active sites. One key structural
difference between the NOS isoforms is the amino acid composition
of the pterin cofactor binding site that is adjacent to the NOS active
site. Previously, we demonstrated that a NOS inhibitor targeting both
the active and pterin sites was potent and functioned as an antimicrobial
(Holden, , Proc. Natl. Acad.
Sci. U.S.A.2013, 110, 1812724145412). Here we present additional crystal structures, binding
analyses, and bacterial killing studies of inhibitors that target
both the active and pterin sites of a bNOS and function as antimicrobials.
Together, these data provide a framework for continued development
of bNOS inhibitors, as each molecule represents an excellent chemical
scaffold for the design of isoform selective bNOS inhibitors.
Fatty acid synthase (FASN), the enzyme
responsible for de novo
synthesis of free fatty acids, is up-regulated in many cancers. FASN
is essential for cancer cell survival and contributes to drug resistance
and poor prognosis. However, it is not expressed in most nonlipogenic
normal tissues. Thus, FASN is a desirable target for drug discovery.
Although different FASN inhibitors have been identified, none has
successfully moved into clinical use. In this study, using in silico
screening of an FDA-approved drug database, we identified proton pump
inhibitors (PPIs) as effective inhibitors of the thioesterase activity
of human FASN. Further investigation showed that PPIs inhibited proliferation
and induced apoptosis of cancer cells. Supplementation of palmitate,
the end product of FASN catalysis, rescued cancer cells from PPI-induced
cell death. These findings provide new evidence for the mechanism
by which this FDA-approved class of compounds may be acting on cancer
The Escherichia coli (E. coli) AlkB protein and its functional human homologues belong to a subfamily of 2-oxoglutarate (2OG)-dependent oxygenases (2OG oxygenases for simplicity) that enable the repair of cytotoxic methylation damage in nucleic acids and which catalyse t-RNA oxidations. DNA alkylation is a major mechanism of action for cytotoxic anti-cancer drugs. Thus, the inhibition of oxidative demethylation, catalyzed by these enzymes, has the potential to improve the efficacy of chemotherapies. Here we report that oligonucleotide aptamers constitute a new class of potent inhibitors of 2OG oxygenases. DNA aptamers can selectively bind to AlkB, with nanomolar affinity, and efficiently inhibit catalysis. The mechanism of inhibition was studied by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Inhibition constants of the aptamers were determined and shown to correlate well with Kd values. The results of kinetic analyses imply that the aptamers bind AlkB away from the active site. Our findings should stimulate the development of oligonucleotide aptamers for human homologues of AlkB and further their study as potential enhancers of chemotherapy efficiency.
A disodium phosphonooxymethyl
prodrug of the antitumor agent triptolide
was prepared from the natural product in three steps (39% yield) and
displayed excellent aqueous solubility at pH 7.4 (61 mg/mL) compared
to the natural product (17 μg/mL). The estimated shelf life
(t90) for hydrolysis of the prodrug at
4 °C and pH 7.4 was found to be two years. In a mouse model of
human colon adenocarcinoma (HT-29), the prodrug administered intraperitoneally
was effective in reducing or eliminating xenograft tumors at dose
levels as low as 0.3 mg/kg when given daily and at 0.9 mg/kg when
given less frequently. When given via intraperitoneal and oral routes
at daily doses of 0.6 and 0.9 mg/kg, the prodrug was also effective
and well tolerated in a mouse model of human ovarian cancer (A2780).
of the MDM2–p53 protein–protein interaction
is being actively pursued as a new anticancer therapeutic strategy,
and spiro-oxindoles have been designed as a class of potent and efficacious
small-molecule inhibitors of this interaction (MDM2 inhibitors). Our
previous study showed that some of our first-generation spiro-oxindoles
undergo a reversible ring-opening-cyclization reaction that, from
a single compound in protic solution, results in an equilibrium mixture
of four diastereoisomers. By exploiting the ring-opening-cyclization
reaction mechanism, we have designed and synthesized a series of second-generation
spiro-oxindoles with symmetrical pyrrolidine C2 substitution. These
compounds undergo a rapid and irreversible conversion to a single,
stable diastereoisomer. Our study has yielded compound 31 (MI-1061), which binds to MDM2 with Ki = 0.16 nM, shows excellent chemical stability, and achieves
tumor regression in the SJSA-1 xenograft tumor model in mice.
Earlier we reported the discovery and design of NBD-556 and their analogs which demonstrated their potential as HIV-1 entry inhibitors. However, progress in developing these inhibitors has been stymied by their CD4-agonist properties, an unfavorable trait for use as drug. Here, we demonstrate the successful conversion of a full CD4-agonist (NBD-556) through a partial CD4-agonist (NBD-09027), to a full CD4-antagonist (NBD-11021) by structure-based modification of the critical oxalamide midregion, previously thought to be intolerant of modification. NBD-11021 showed unprecedented neutralization breath for this class of inhibitors, with pan-neutralization against a panel of 56 Env-pseudotyped HIV-1 representing diverse subtypes of clinical isolates (IC50 as low as 270 nM). The cocrystal structure of NBD-11021 complexed to a monomeric HIV-1 gp120 core revealed its detail binding characteristics. The study is expected to provide a framework for further development of NBD series as HIV-1 entry inhibitors for clinical application against AIDS.
Compstatin peptides are complement
inhibitors that bind and inhibit
cleavage of complement C3. Peptide binding is enhanced by hydrophobic
interactions; however, poor solubility promotes aggregation in aqueous
environments. We have designed new compstatin peptides derived from
the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized
between C2 and C12), based on structural, computational, and experimental
studies. Furthermore, we developed and utilized a computational framework
for the design of peptides containing non-natural amino acids. These
new compstatin peptides contain polar N-terminal extensions and non-natural
amino acid substitutions at positions 4 and 9. Peptides with α-modified
non-natural alanine analogs at position 9, as well as peptides containing
only N-terminal polar extensions, exhibited similar activity compared
to W4A9, as quantified via ELISA, hemolytic, and cell-based assays,
and showed improved solubility, as measured by UV absorbance and reverse-phase
HPLC experiments. Because of their potency and solubility, these peptides
are promising candidates for therapeutic development in numerous complement-mediated
The JmjC oxygenases catalyse the N-demethylation of Nε-methyl lysine residues in histones and are current therapeutic targets. A set of 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.