Inhibition of a viral enzyme by a small-molecule dimer disruptor.

We identified small-molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi's sarcoma-associated herpesvirus (KSHV) by screening an alpha-helical mimetic library. Next, we synthesized a second generation of low-micromolar inhibitors with improved potency and solubility. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor. 1H-15N HSQC titration studies mapped the inhibitor binding site to the dimer interface, and mutagenesis studies targeting this region were consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of a dynamic intermediate. These results validate the interface of herpesvirus proteases and other similar oligomeric interactions as suitable targets for the development of small-molecule inhibitors

Oxazole and thiazole analogs of sulindac for cancer prevention

Aim: Experimental and epidemiological studies and clinical trials suggest that nonsteroidal anti-inflammatory drugs possess antitumor potential. Sulindac, a widely used nonsteroidal anti-inflammatory drug, can prevent adenomatous colorectal polyps and colon cancer, especially in patients with familial adenomatous polyposis. Sulindac sulfide amide (SSA) is an amide-linked sulindac sulfide analog that showed in vivo antitumor activity in a human colon tumor xenograft model. Results/methodology: A new analog series with heterocyclic rings such as oxazole or thiazole at the C-2 position of sulindac was prepared and screened against prostate, colon and breast cancer cell lines to probe the effect of these novel substitutions on the activity of sulindac analogs.Conclusion: In general, replacement of the amide function of SSA analogs had a negative impact on the cell lines tested. A small number of hits incorporating rigid oxazole or thiazole groups in the sulindac scaffold in place of the amide linkage show comparable activity to our lead agent SSA.</p

Diverse amide analogs of sulindac for cancer treatment and prevention

Sulindac is a non-steroidal anti-inflammatory drug (NSAID) that has shown significant anticancer activity. Sulindac sulfide amide (1) possessing greatly reduced COX-related inhibition relative to sulindac displayed in vivoantitumor activity that was comparable to sulindac in a human colon tumorxenograft model. Inspired by these observations, a panel of diverse sulindac amide derivatives have been synthesized and their activity probed against three cancer cell lines (prostate, colon and breast). A neutral analog, compound 79 was identified with comparable potency relative to lead 1 and activity against a panel of lymphoblastic leukemia cell lines. Several new series also show good activity relative to the parent (1), including five analogs that also possess nanomolar inhibitory potencies against acute lymphoblastic leukemia cells. Several new analogs identified may serve as anticancer lead candidates for further development

Screening and development of new inhibitors of FtsZ from <i>M. Tuberculosis</i>

A variety of commercial analogs and a newer series of Sulindac derivatives were screened for inhibition of M. tuberculosis (Mtb) in vitro and specifically as inhibitors of the essential mycobacterial tubulin homolog, FtsZ. Due to the ease of preparing diverse analogs and a favorable in vivo pharmacokinetic and toxicity profile of a representative analog, the Sulindac scaffold may be useful for further development against Mtb with respect to in vitro bacterial growth inhibition and selective activity for Mtb FtsZ versus mammalian tubulin. Further discovery efforts will require separating reported mammalian cell activity from both antibacterial activity and inhibition of Mtb FtsZ. Modeling studies suggest that these analogs bind in a specific region of the Mtb FtsZ polymer that differs from human tubulin and, in combination with a pharmacophore model presented herein, future hybrid analogs of the reported active molecules that more efficiently bind in this pocket may improve antibacterial activity while improving other drug characteristics

Role of electrostatic interactions in PDZ domain ligand recognition

ABSTRACT: PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse R1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements. Cells rely on modular protein-protein recognition domains to assemble multiprotein signaling complexes. PDZ 1 (PSD-95, Dlg, ZO-1 homology) domains form a large family of such modules. PDZ domain-containing proteins play an important role in organizing signaling structures at cellcell signaling junctions such as synapses (1, 2). PDZ domains were first characterized for their ability to specifically recognize C-terminal peptide ligands, including the C-termini of receptors and channels Here we describe experiments aimed at more precisely evaluating the role of electrostatic interactions in PDZmediated peptide recognition using the Mus musculus R1-syntrophin PDZ domain as a model system. First, we have determined the sensitivity of various PDZ-mediated interactions to ionic strength, and second, we have determined the effects of mutating a conserved basic residue in the PDZ binding groove that has been proposed to play a role in electrostatic recognition. These results indicate that electrostatics do not play a significant role in recognition of the terminal carboxylate. In addition, we have examined a library of ligand variants and found that PDZ domains discriminate very tightly between ligands with minor alterations in the precise chemical structure of the C-terminus. Together, these results are consistent with a model in which steric and † This research was supported by grants from the National Institutes of Health and the David and Lucille Packard Foundation (to W.A.L.) and the Stewart Trust (to R.K.G. alcohol; Fmoc, 9-fluorenylmethoxycarbonyl; FPLC, fast performance liquid chromatography; HATU, 2-(1H-9-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HPLC, high-pressure liquid chromatography; IPTG, isopropyl -D-thiogalactopyranoside; MeOH, methanol or methyl alcohol; Ni-NTA, nickel nitriloacetic acid; nNOS, neuronal nitric oxide synthase; PCR, polymerase chain reaction; PDZ, PSD-95, Dlg, ZO-1 homology; TEV, tobacco etch virus; TFA, trifluoroacetic acid; TIS, triisopropylsilane

Cysteine Modifiers Suggest an Allosteric Inhibitory Site on the CAL PDZ Domain

Protein–protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that plays important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of the PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and suggests an allosteric connection between target residue Cys319 and the conserved Leu291 in the GLGI motif. MD and ED thus appear to represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action may exploit the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein–protein binding modules

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription

Anticancer properties of distinct antimalarial drug classes

We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC 50 s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC 50 s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings

Selecting an Anti-Malarial Clinical Candidate from Two Potent Dihydroisoquinolones

BACKGROUND: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described. METHODS: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs. RESULTS: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg. CONCLUSION: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators

Similarly Efficacious Anti-Malarial Drugs SJ733 and Pyronaridine Differ in Their Ability to Remove Circulating Parasites in Mice

BACKGROUND: Artemisinin-based combination therapy (ACT) has been a mainstay for malaria prevention and treatment. However, emergence of drug resistance has incentivised development of new drugs. Defining the kinetics with which circulating parasitized red blood cells (pRBC) are lost after drug treatment, referred to as the parasite clearance curve , has been critical for assessing drug efficacy; yet underlying mechanisms remain partly unresolved. The clearance curve may be shaped both by the rate at which drugs kill parasites, and the rate at which drug-affected parasites are removed from circulation. METHODS: In this context, two anti-malarials, SJ733, and an ACT partner drug, pyronaridine were compared against sodium artesunate in mice infected with Plasmodium berghei (strain ANKA). To measure each compound\u27s capacity for pRBC removal in vivo, flow cytometric monitoring of a single cohort of fluorescently-labelled pRBC was employed, and combined with ex vivo parasite culture to assess parasite maturation and replication. RESULTS: These three compounds were found to be similarly efficacious in controlling established infection by reducing overall parasitaemia. While sodium artesunate acted relatively consistently across the life-stages, single-dose SJ733 elicited a biphasic effect, triggering rapid, partly phagocyte-dependent removal of trophozoites and schizonts, followed by arrest of residual ring-stages. In contrast, pyronaridine abrogated maturation of younger parasites, with less pronounced effects on mature parasites, while modestly increasing pRBC removal. CONCLUSIONS: Anti-malarials SJ733 and pyronaridine, though similarly efficacious in reducing overall parasitaemia in mice, differed markedly in their capacity to arrest replication and remove pRBC from circulation. Thus, similar parasite clearance curves can result for anti-malarials with distinct capacities to inhibit, kill and clear parasites