A series of four stable synthetic bacteriochlorins was tested in vitro in HeLa cells for their potential in photodynamic therapy (PDT). The parent bacteriochlorin (BC), dicyano derivative (NC)2BC and corresponding zinc chelate (NC)2BC–Zn and palladium chelate (NC)2BC–Pd were studied. Direct dilution of a solution of bacteriochlorin in an organic solvent (N,N-dimethylacetamide) into serum-containing medium was compared with the dilution of bacteriochlorin in Cremophor EL (CrEL; polyoxyethylene glycerol triricinoleate) micelles into the same medium. CrEL generally reduced aggregation (as indicated by absorption and fluorescence) and increased activity up to tenfold (depending on bacteriochlorin), although it decreased cellular uptake. The order of PDT activity against HeLa human cancer cells after 24 h incubation and illumination with 10 J cm−2 of near-infrared (NIR) light is (NC)2BC–Pd (LD50 = 25 nm) > (NC)2BC > (NC)2BC–Zn ≈ BC. Subcellular localization was determined to be in the endoplasmic reticulum, mitochondria and lysosomes, depending on the bacteriochlorin. (NC)2BC–Pd showed PDT-mediated damage to mitochondria and lysosomes, and the greatest production of hydroxyl radicals as determined using a hydroxyphenylfluorescein probe. The incorporation of cyano substituents provides an excellent motif for the enhancement of the photoactivity and photostability of bacteriochlorins as PDT photosensitizers.
antitumor agents; Cremophor EL micelles; photodynamic therapy; photostability; reactive oxygen species; synthetic bacteriochlorins
Monoamine Transporters; Triple reuptake inhibitors; Pyran; Antidepressants
Successful Influenza A viral replication requires both viral proteins and host cellular factors. Here we utilized a cellular assay to screen for small molecules capable of interfering with any of such necessary viral or cellular components. We employed an established reporter assay assessing influenza viral replication by monitoring the activity of co-expressed luciferase. We screened a diverse chemical compound library, resulting in the identification of compound 7, inhibiting a novel yet elusive target. Quantitative real-time PCR studies confirmed the dose dependent inhibitory activity of compound 7 in a viral replication assay. Furthermore, we showed that compound 7 was effective in rescuing high dose influenza infection in an in vivo mouse model. As oseltamivir-resistant influenza strains emerge, compound 7 could be further investigated as a possible novel scaffold for the development of anti-influenza agents acting on novel targets.
Influenza virus; Drug discovery; Ugi reaction; tetrazole formation
Using a pyrrole-based scaffold, we developed a series of small molecules that mimic the three-dimensional arrangement of the polar and hydrophobic functional groups of the best cyclic-peptide inhibitor. Iterative optimization cycles of design, synthesis and kinetic testing has lead to an effective inhibitor of Wip1, that is selective for this phosphatase over others. The picture shows the structure of the best inhibitor bound to the active site of the enzyme.
Wip1; Inhibitor; Small Molecule
drug design; drug discovery; carbazoles; lansine; leishmaniasis
A series of (±)-6-alkyl-2,4-diaminopyrimidine-based inhibitors of bacterial dihydrofolate reductase (DHFR) have been prepared and evaluated for biological potency against Bacillus anthracis and Staphylococcus aureus. Biological studies reveal attenuated activity relative to earlier structures lacking substitution at C6 of the diaminopyrimidine moiety, though minimum inhibitory concentration (MIC) values are in the 0.125–8 μg/mL range for both organisms. This effect was rationalized from previous three-dimensional X-ray structure studies that indicate the presence of a side pocket containing two water molecules adjacent to the main binding pocket. Because of the hydrophobic nature of the substitutions at C6 the main interactions are with protein residues Leu20 and Leu28. These interactions lead to a minor conformational change in the protein, which opens the pocket containing these waters such that it is continuous with the main binding pocket. These water molecules are reported to play a critical role in the catalytic reaction. This highlights a new area for inhibitor expansion within the limited architectural variation at the catalytic site of bacterial DHFR.
6-Alkylpyrimidine-based antibiotics; DHFR inhibitors; Bacillus anthracis; Staphylococcus aureus
Ca2+-activated K+ channels (KCa) play a pivotal role in the physiology of a wide variety of tissues and disease states, including vascular endothelia, secretory epithelia, certain cancers, red blood cells (RBC), neurons and immune cells. Such widespread involvement has generated an intense interest in elucidating the function and regulation of these channels, with the goal of developing pharmacological strategies aimed at selective modulation of KCa channels in various disease states. Herein, we give an overview of the molecular and functional properties of these channels and their therapeutic importance as well as discuss the achievements made in designing pharmacological tools which control the function of KCa channels by modulating their gating properties. Moreover, this review discusses the recent advances in our understanding of KCa channel assembly and anterograde trafficking toward the plasma membrane, the microdomains in which these channels are expressed within the cell and finally the retrograde trafficking routes these channels take following endocytosis. As both the regulation of intracellular trafficking by agonists, as well as the protein-protein interactions that modify these events continue to be explored, we anticipate this will open up new therapeutic avenues for the targeting of these channels based on the pharmacological modulation of KCa channel density at the plasma membrane.
KCa3.1; KCa2.1; KCa2.2; KCa2.3; channel density; trafficking; pharmacological modulators
multicomponent reaction; drug discovery; protein-protein interaction; p53-Mdm2; fluorine
glucagon-like peptide-1 (GLP-1); human pancreatic islets; insulinotropic effects; receptor agonists; vitamin B12
The synthesis of halogenated analogs of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (1), known commonly as bexarotene, and their evaluation for retinoid-X-receptor (RXR)-specific agonist performance is described. Compound 1 is FDA approved to treat cutaneous T-cell lymphoma (CTCL); however, bexarotene treatment can induce hypothyroidism and elevated triglyceride levels, presumably by disrupting RXR heterodimer pathways for other nuclear receptors. The novel halogenated analogs in this study were modeled and assessed for their ability to bind to RXR and stimulate RXR homodimerization in an RXRE-mediated transcriptional assay as well as an RXR mammalian-2-hybrid assay. In an array of 8 novel compounds, 4 analogs were discovered to promote RXR-mediated transcription with comparable EC50 values as 1 and are selective RXR agonists. Our approach also uncovered a periodic trend of increased binding and homodimerization of RXR when substituting a halogen atom for a proton ortho to the carboxylic acid on 1.
Bexarotene; Cutaneous T Cell Lymphoma; Retinoic Acid Receptor; Retinoid X Receptor; Rexinoid
Microtubule stabilizers are powerful anti-mitotic compounds and represent a proven cancer treatment strategy. Several classes of compounds in clinical use or trials, such as the taxanes and epothilones, bind to the same region of β-tubulin. Determining how these molecules interact with tubulin and stabilize microtubules is important both for understanding the mechanism of action and enhancing chemotherapeutic potential, e.g. reducing side effects, increasing solubility, and overcoming resistance. Structural studies using nonpolymerized tubulin or stabilized polymers have produced different models of epothilone binding. Here, we used directed mutagenesis of the binding site on Saccharomyces cerevisiae β-tubulin to analyze interactions between Epothilone B and its biologically relevant substrate, dynamic microtubules. Five engineered amino acid changes contributed to a 125-fold increase in Epothilone B cytotoxicity independent of inherent microtubule stability. The mutagenesis of endogenous β-tubulin was done in otherwise isogenic strains. This facilitated the correlation of amino acid substitutions with altered cytotoxicity using molecular mechanics simulations. The results, which are based on the interaction between Epothilone B and dynamic microtubules, most strongly support the binding mode determined by NMR spectroscopy-based studies. This work establishes a system for discriminating between potential binding modes and among various compounds and/or analogues using a sensitive biological activity-based readout.
epothilone; microtubule; tubulin; taxol binding site; microtubule stabilizer; drug design; antitumor agents
flavonoid; naringenin; resveratrol; abyssinone II; antitubercular; antibacterial mechanistic study
The interaction of CXCR4 with CXCL12 (SDF-1) plays a critical role in cancer metastasis by facilitating the homing of tumor cells to metastatic sites. Based on our previously published work on CXCR4 antagonists, we have synthesized a series of aryl sulfonamides that inhibit the CXCR4/CXCL12 interaction. Analog bioactivities were assessed with binding affinity and Matrigel invasion assays. Computer modeling was employed to evaluate a selection of the new analogs docked into the CXCR4 X-ray structure and to rationalize discrepancies between the affinity and Matrigel in vitro assays. A lead compound 5a displays subnanomolar potency in the binding affinity assay (IC50 = 8.0 nM) and the Matrigel invasion assay (100% blockade of invasion at 10 nM). These data demonstrate that benzenesulfonamides are a unique class of CXCR4 antagonists with high potency.
CXCR4 inhibitors; metastasis; sulfonamides; inflammation
Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be improved by increasing the molecular complexity, by inserting groups able to establish additional interaction with the binding pocket of the enzyme. This strategy has been pursued with the synthesis of α-amino-substituted analogues of Acivicin and N1-substituted-pyrazoline derivatives. In general, there is a direct correlation between the enzymatic activity and the in vitro anti-trypanosomal efficacy of the derivatives studied here. However, this cannot be taken as a general rule, since other important factors may play a role, notably the ability of uptake / diffusion of the molecules into the trypanosomes.
CTP synthetase; Trypanosoma; Amino acid; Isoxazoline; Pyrazoline
Pathogenicity of Yersinia pestis (Y. pestis) relies on several effector proteins, including YopH, a protein-tyrosine phosphatase. Previously, we screened a library of analogues based on the ubiquitous PTP substrate, p-nitrophenylphosphate (pNPP) and found that incorporation of a 3-phenyl substituent (6-nitro-[1,1'-biphenyl]-3-yl dihydrogen phosphate (1)) enhanced affinity. The current study reports the conversion of 1 from a substrate to inhibitor by replacing the hydrolysable phosphoryl group with a 3-isoxazolecarboxylic acid moiety and by introduction of an aminooxy group and subsequent diversification using oxime-based click chemistry. As reported herein, this approach led to the identification of non-promiscuous low micromolar affinity bidentate YopH inhibitors.
Substrate screening; Aminooxy platform; Oxime-based click chemistry; YopH inhibitors; In silico docking studies
Can we consider cancer as a “metabolic disease”? Tumors are the result of a metabolic selection, forming tissues composed of heterogeneous cells that generally express an overactive metabolism as a common feature. In fact, cancer cells have to deal with increased needs for both energy and biosynthetic intermediates, in order to support their growth and invasiveness. However, their high proliferation rate often generates regions that are not sufficiently oxygenated. Therefore, their carbohydrate metabolism has to rely mostly on a glycolytic process that is uncoupled from oxidative phosphorylation. This metabolic switch, also known as the “Warburg Effect”, constitutes a fundamental adaptation of the tumor cells to a relatively hostile environment, and supports the evolution of aggressive and metastatic phenotypes. As a result, tumor glycolysis may constitute an attractive target for cancer therapy. This approach has often raised concerns that anti-glycolytic agents may cause serious side effects on normal cells. Actually, the key for a selective action against cancer cells can be found in their hyperbolic addiction to glycolysis, which may be exploited to generate new anti-cancer drugs showing minimal toxicity. In fact, there is growing evidence that supports many glycolytic enzymes and transporters as suitable candidate targets for cancer therapy. Herein we review some of the most relevant anti-glycolytic agents that have been investigated so far for the treatment of cancer.
anticancer agents; glycolysis; inhibitors; tumor metabolism; warburg effect
antibiotic; Bacteroides fragilis; ribonuclease; metronidazole; prodrug
To discover more potent p97 inhibitors, we have carried out a structure-activity relationship study of the quinazoline scaffold previously identified from our HTS campaigns. Two improved inhibitors, ML240 and ML241, inhibited p97 ATPase with IC50s of 100 nm. Both compounds inhibited degradation of a p97-dependent but not a p97-independent proteasome substrate in a dual-reporter cell line. They also impaired the endoplasmic reticulum associated degradation (ERAD) pathway. Unexpectedly, ML240 potently stimulated accumulation of LC3-II within minutes, inhibited cancer cell growth, and rapidly mobilized the executioner caspases 3 and 7 whereas ML241 did not. The behavior of ML240 suggests that disruption of the protein homeostasis function of p97 leads to more rapid activation of apoptosis than is observed with a proteasome inhibitor. Further characterization revealed that ML240 has broad anti-proliferative activity towards the NCI-60 panel of cancer cell lines but slightly lower activity towards normal cells. ML240 also synergizes with the proteasome inhibitor MG132 to kill multiple colon cancer cell lines. Meanwhile, both probes have low off-target activity towards a panel of protein kinases and central nervous system targets. Our results nominate ML240 as a promising starting point for the development of a novel agent for chemotherapy of cancer, and provide a rationale for developing pathway-specific p97 inhibitors.
AAA ATPase; autophagy; cancer; structure-activity relationships; ubiquitin proteasome system
Human African trypanosomiasis (HAT) is a life-threatening disease with approximately 30 000–40 000 new cases each year. Trypanosoma brucei protein kinase GSK3 short (TbGSK3) is required for parasite growth and survival. Herein we report a screen of a focused kinase library against T. brucei GSK3. From this we identified a series of several highly ligand-efficient TbGSK3 inhibitors. Following the hit validation process, we optimised a series of diaminothiazoles, identifying low-nanomolar inhibitors of TbGSK3 that are potent in vitro inhibitors of T. brucei proliferation. We show that the TbGSK3 pharmacophore overlaps with that of one or more additional molecular targets.
antiprotozoal agents; GSK3; medicinal chemistry; protein kinases; Trypanosoma brucei
The emergence of virulent, drug-resistant bacterial strains coupled with a minimal output of new pharmaceutical agents to combat them makes this a critical time for antibacterial research. Aminoglycosides are a well-studied, highly potent class of naturally occurring antibiotics with scaffolds amenable to modification, and therefore, they provide an excellent starting point for the development of semisynthetic, next-generation compounds. To explore the potential of this approach, we synthesized a small library of aminoglycoside derivatives selectively and minimally modified at one or two positions with a guanidine group replacing the corresponding amine or hydroxy functionality. Most guanidino-aminoglycosides showed increased affinity for the ribosomal decoding rRNA site, the cognate biological target of the natural products, when compared with their parent antibiotics, as measured by an in vitro fluorescence resonance energy transfer (FRET) A-site binding assay. Additionally, certain analogues showed improved minimum inhibitory concentration (MIC) values against resistant bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA). An amikacin derivative holds particular promise with activity greater than or equal to the parent antibiotic in the majority of bacterial strains tested.
amikacin; aminoglycosides; antibiotics; A-site RNA; methicillin-resistant Staphylococcus aureus (MRSA)
The HIV-1 Vif protein, essential for in vivo viral replication, protects the virus from innate antiviral cellular factor APOBEC3G (A3G), and is an attractive target for developing antiviral therapeutics. Here we have evaluated the structure-activity relationships of RN18, a small molecule recently identified as an inhibitor of Vif function that blocks viral replication only in non-permissive cells expressing A3G, by inhibiting Vif-A3G interactions. Microwave-assisted cross-coupling reactions were developed to prepare a series of RN18 analogues with diverse linkages and substitutions on the phenyl rings. A dual cell-based assay system was used to assess antiviral activity against wild-type HIV-1 in both non-permissive (H9) and permissive (MT-4) cells that also allowed evaluation of specificity. In general, variations of phenyl substitutions were detrimental for antiviral potency and specificity, but isosteric replacements of amide and ether linkages were relatively well tolerated. These SAR data define structural requirements for Vif-specific activity, identify new compounds with improved antiviral potency and specificity, and provide leads for further exploration to develop new antiviral therapeutics.
APOBEC3G; HIV-1 Vif; inhibitors; structure-activity relationships; drug discovery; antiviral agents
CK-666 (1) is a recently discovered small molecule inhibitor of the Arp2/3 complex, a key actin cytoskeleton regulator with roles in bacterial pathogenesis and motility of cancer cells. While 1 is commercially available, the crystal structure of Arp2/3 (Actin-related protein 2/3) complex with 1 bound has not been reported, making its mechanism of action uncertain. Furthermore, its relatively low potency increases its potential for off target effects in vivo, complicating interpretation of its influence in cell biological studies and precluding its use in clinical applications. Here we report the crystal structure of 1 bound to Arp2/3 complex, which reveals that 1 binds between the Arp2 and Arp3 subunits to stabilize the inactive conformation of the complex. Based on the crystal structure, we used computational docking and free energy perturbation calculations of monosubstituted derivatives of 1 to guide optimization efforts. Biochemical assays of ten newly synthesized compounds led to the identification of compound 2, which exhibits a 3 fold increase in inhibitory activity in vitro. In addition, our computational analyses unveiled a surface groove at the interface of the Arp2 and Arp3 subunits that can be exploited for additional structure-based optimization.
Actin; Arp2/3 complex; Amides; Cancer; Chlorine; Free Energy Perturbation; Polymers
The Eph–ephrin system, including the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist able to inhibit EphA2 receptor activation and therefore potentially useful as a novel EphA2 receptor targeting agent. Here, we explore the structure-activity relationships of a focused set of lithocholic acid derivatives, based on molecular modelling investigation and displacement binding assays. Our exploration shows that while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand-binding domain of EphA2, with a steady-state dissociation constant (KD) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 and cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrin-A1 ligand. These findings suggest that cholanic acid can be used as a template structure to design effective EphA2 antagonists, with potential impact in the elucidation of the role played by this receptor in pathological conditions.
Protein-protein interactions; Structure-activity relationships; Surface plasmon resonance; Steroids; Drug design
A series of synthesized and commercially available compounds were assessed against PI3Kα
for in vitro inhibitory activity and the results compared to binding calculated in silico. Using
published crystal structures of PI3Kγ and PI3Kδ co-crystallized with inhibitors as a
template, docking was able to identify the majority of potent inhibitors from a decoy set of 1000
compounds. On the other hand, PI3Kα in the apo-form, modeled by induced fit docking, or built
as a homology model gave only poor results. A PI3Kα homology model derived from a
ligand-bound PI3Kδ crystal structure was developed that has a good ability to identify active
compounds. The docking results identified binding poses for active compounds that differ from those
identified to date and can contribute to our understanding of structure–activity
relationships for PI3K inhibitors.
homology modeling; inhibitors; PI3K kinases; rhodanine; thiazolidinedione
The ability of bacteria expressing inhibitor resistant (IR) β-lactamases is stimulating the development of novel inhibitors. SA2-13 was previously designed to enhance the stabilization of the deacylation refractory, trans-enamine inhibitory intermediate. To test whether this mode of inhibition can overcome different IR mutations, we determined the binding mode of SA2-13 after soaking the inhibitor into crystals of the IR SHV β-lactamase variants S130G and M69V. The 1.45Å crystal structure of the S130G SHV: SA2-13 complex reveals that SA2-13 is still able to form the stable trans-enamine intermediate similar to the wild type complex structure yet with its carboxyl linker shifted deeper into the active site in the space vacated by the S130G mutation. In contrast, data from crystals of the M69V SHV variant soaked with SA2-13 at 1.3 Å did not reveal clear inhibitor density indicating that this IR variant disfavors the trans-enamine conformation, likely due to a subtle shift in A237.
β-lactamase inhibitor; protein crystallography