Development of a Novel DNA Mono-alkylator Platform for Antibody-Drug Conjugates.

Although microtubule inhibitors (MTI) remain a therapeutically valuable payload option for antibody-drug conjugates (ADC), some cancers do not respond to MTI-based ADCs. Efforts to fill this therapeutic gap have led to a recent expansion of the ADC payload "toolbox" to include payloads with novel mechanisms of action such as topoisomerase inhibition and DNA cross-linking. We present here the development of a novel DNA mono-alkylator ADC platform that exhibits sustained tumor growth suppression at single doses in MTI-resistant tumors and is well tolerated in the rat upon repeat dosing. A phosphoramidate prodrug of the payload enables low ADC aggregation even at drug-to-antibody ratios of 5:1 while still delivering a bystander-capable payload that is effective in multidrug resistant (MDR)-overexpressing cell lines. The platform was comparable in xenograft studies to the clinical benchmark DNA mono-alkylator ADC platform DGN459 but with a significantly better tolerability profile in rats. Thus, the activity and tolerability profile of this new platform make it a viable option for the development of ADCs.

Discovery and Optimization of a STING Agonist Platform for Application in Antibody Drug Conjugates.

While STING agonists have proven to be effective preclinically as anti-tumor agents, these promising results have yet to be translated in the clinic. A STING agonist antibody-drug conjugate (ADC) could overcome current limitations by improving tumor accessibility, allowing for systemic administration as well as tumor-localized activation of STING for greater anti-tumor activity and better tolerability. In line with this effort, a STING agonist ADC platform was identified through systematic optimization of the payload, linker, and scaffold based on multiple factors including potency and specificity in both in vitro and in vivo evaluations. The platform employs a potent non-cyclic dinucleotide STING agonist, a cleavable ester-based linker, and a hydrophilic PEG8-bisglucamine scaffold. A tumor-targeted ADC built with the resulting STING agonist platform induced robust and durable anti-tumor activity and demonstrated high stability and favorable pharmacokinetics in nonclinical species.

Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates.

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10-11-10-8 M) against a variety of HER2-expressing cancer cell lines. Several peptide-linked variants of the lead compound were prepared and conjugated to trastuzumab to afford ADCs with drug-to-antibody (DAR) ratios ranging from 3 to 5. The ADCs exhibited antigen-dependent cytotoxicity in vitro and potently suppressed tumor xenograft growth in vivo in a target-dependent manner. Moreover, the ADCs were well-tolerated in both mouse and rat. This work demonstrates for the first time that PBD polyamide hybrids can serve as effective ADC payloads.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

Changes of cellular stress response related hsp70 and abcb1 transcript and Hsp70 protein levels in Siberian freshwater amphipods upon exposure to cadmium chloride in the lethal concentration range.

The induction of cellular stress response systems, heat shock protein hsp70/Hsp70 and multixenobiotic transporter abcb1, by cadmium chloride (CdCl2) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal concentrations (LC) of CdCl2, the sensitivities of the different species to CdCl2 were ranked (24 hr LC50 in mg/L CdCl2 (mean/95% confidence interval)): Gammarus lacustris (1.7/1.3-2.4) < Eulimnogammarus cyaneus (2.9/2.1-4.0) < Eulimnogammarus verrucosus (5.7/3.8-8.7) < Eulimnogammarus vittatus (18.1/12.4-26.6). Conjugated dienes, indicating lipid peroxidation, were significantly increased after 24 hr exposures to 5 mg/L CdCl2 only in the more CdCl2-sensitive species G. lacustris and E. cyaneus. Upon treatment with 0.54 to 5.8 mg/L CdCl2 for 1, 6 and 24 hrs, hsp70 transcript levels were generally more increased after the longer exposure times and in the more CdCl2-sensitive species. Relating the CdCl2 exposure concentrations to LCx values revealed that across the species the increases of hsp70 transcript levels were comparatively low (up to 2.6-fold) at CdCl2 concentrations ≤LC50. Relative hsp70 transcript levels were maximally increased in E. cyaneus by 5 mg/L CdCl2 ( = ˆ LC70) at 24 hrs (9.1-fold increase above the respective control). When G. lacustris was exposed to 5 mg/L CdCl2 ( = ˆ LC90) for 24 hrs, the increase in hsp70 was in comparison to E. cyaneus considerably less pronounced (3.0-fold increase in hsp70 levels relative to control). Upon exposure of amphipods to 5 mg/L CdCl2, increases in Hsp70 protein levels compared to untreated controls were highest in E. cyaneus at 1 and 6 hrs (5 mg/L CdCl2 = ˆ LC70) and in E. verrucosus at 24 hrs (5 mg/L CdCl2 = ˆ LC45). Thus, when the fold increases in Hsp70 protein levels in the different amphipod species were related to the respective species-specific LCx values a similar bell-shaped trend as for hsp70 transcript levels was seen across the species. Transcript levels of abcb1 in CdCl2exposed individuals of the different amphipod species varied up to 4.7-fold in relation to the respective controls. In contrast to hsp70/Hsp70, abcb1 transcripts in CdCl2 exposed individuals of the different amphipod species did not indicate similar levels of induction of abcb1 at equal LCx levels across the species. Induction of hsp70 and abcb1 genes and Hsp70 proteins by CdCl2 in the lethal concentration range shows that these cellular responses are rather insensitive to CdCl2 stress in the examined amphipod species. Furthermore, the increase of expression of these cellular defense systems at such high stress levels suggests that induction of these genes is not related to the maintenance of normal metabolism but to mitigation of the effects of severe toxic stress.

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models.

PURPOSE: To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. EXPERIMENTAL DESIGN: Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. RESULTS: Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a "hypoxia index" compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. CONCLUSIONS: These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins.

Antileukemia activity of the novel peptidic CXCR4 antagonist LY2510924 as monotherapy and in combination with chemotherapy.

Targeting the stromal cell-derived factor 1α (SDF-1α)/C-X-C chemokine receptor type 4 (CXCR4) axis has been shown to be a promising therapeutic approach to overcome chemoresistance in acute myeloid leukemia (AML). We investigated the antileukemia efficacy of a novel peptidic CXCR4 antagonist, LY2510924, in preclinical models of AML. LY2510924 rapidly and durably blocked surface CXCR4 and inhibited stromal cell-derived factor 1 (SDF-1)α-induced chemotaxis and prosurvival signals of AML cells at nanomolar concentrations more effectively than the small-molecule CXCR4 antagonist AMD3100. In vitro, LY2510924 chiefly inhibited the proliferation of AML cells with little induction of cell death and reduced protection against chemotherapy by stromal cells. In mice with established AML, LY2510924 caused initial mobilization of leukemic cells into the circulation followed by reduction in total tumor burden. LY2510924 had antileukemia effects as monotherapy as well as in combination with chemotherapy. Gene expression profiling of AML cells isolated from LY2510924-treated mice demonstrated changes consistent with loss of SDF-1α/CXCR4 signaling and suggested reduced proliferation and induction of differentiation, which was proved by showing the attenuation of multiple prosurvival pathways such as PI3K/AKT, MAPK, and ß-catenin and myeloid differentiation in vivo. Effective disruption of the SDF-1α/CXCR4 axis by LY2510924 may translate into effective antileukemia therapy in future clinical applications.

Identification of a putatively multixenobiotic resistance related Abcb1 transporter in amphipod species endemic to the highly pristine Lake Baikal.

The fauna of Lake Baikal in Eastern Siberia, the largest freshwater body on Earth, is characterized by high degrees of biodiversity and endemism. Amphipods, a prominent taxon within the indigenous fauna, occur in an exceptionally high number of endemic species. Considering the specific water chemistry of Lake Baikal with extremely low levels of potentially toxic natural organic compounds, it seems conceivable that certain adaptions to adverse environmental factors are missing in endemic species, such as cellular defense mechanisms mitigating toxic effects of chemicals. The degree to which the endemic fauna is affected by the recently occurring anthropogenic water pollution of Lake Baikal may depend on the existence of such cellular defense mechanisms in those species. We here show that endemic amphipods express transcripts for Abcb1, a major component of the cellular multixenobiotic resistance (MXR) defense against toxic chemicals. Based on a partial abcb1 cDNA sequence from Gammarus lacustris, an amphipod species common across Northern Eurasia but only rarely found in Lake Baikal, respective homologous sequences were cloned from five amphipods endemic to Lake Baikal, Eulimnogammarus verrucosus, E. vittatus, E. cyaneus, E. marituji, and Gmelinoides fasciatus, confirming that abcb1 is transcribed in those species. The effects of thermal (25 °C) and chemical stress (1-2 mg L(-1) phenanthrene) in short-term exposures (up to 24 h) on transcript levels of abcb1 and heat shock protein 70 (hsp70), used as a proxy for cellular stress in the experiments, were exemplarily examined in E. verrucosus, E. cyaneus, and Gammarus lacustris. Whereas increases of abcb1 transcripts upon treatments occurred only in the Baikalian species E. verrucosus and E. cyaneus but not in Gammarus lacustris, changes of hsp70 transcript levels were seen in all three species. At least for species endemic to Lake Baikal, the data thus indicate that regulation of the identified amphipod abcb1 is triggered within the general cellular stress response. This is the first report presenting molecular data on a MXR transporter in amphipods, an ecotoxicologically important but with regard to gene sequence data comparatively little explored taxon.

Identification of SQ609 as a lead compound from a library of dipiperidines.

We recently reported that compounds created around a dipiperidine scaffold demonstrated activity against Mycobacterium tuberculosis (Mtb) (Bogatcheva, E.; Hanrahan, C.; Chen, P.; Gearhart, J.; Sacksteder, K.; Einck, L.; Nacy, C.; Protopopova, M. Bioorg. Med. Chem. Lett.2010, 20, 201). To optimize the dipiperidine compound series and to select a lead compound to advance into preclinical studies, we evaluated the structure-activity relationship (SAR) of our proprietary libraries. The (piperidin-4-ylmethyl)piperidine scaffold was an essential structural element required for antibacterial activity. Based on SAR, we synthesized a focused library of 313 new dipiperidines to delineate additional structural features responsible for antitubercular activity. Thirty new active compounds with MIC 10-20 µg/ml on Mtb were identified, but none was better than the original hits of this series, SQ609, SQ614, and SQ615. In Mtb-infected macrophages in vitro, SQ609 and SQ614 inhibited more than 90% of intracellular bacterial growth at 4 µg/ml; SQ615 was toxic to these cells. In mice infected with Mtb, weight loss was completely prevented by SQ609, but not SQ614, and SQ609 had a prolonged therapeutic effect, extended by 10-15 days, after cessation of therapy. Based on in vitro and in vivo antitubercular activity, SQ609 was identified as the best-in-class dipiperidine compound in the series.

Chemical modification of capuramycins to enhance antibacterial activity.

OBJECTIVES: To extend capuramycin spectrum of activity beyond mycobacteria and improve intracellular drug activity. METHODS: Three capuramycin analogues (SQ997, SQ922 and SQ641) were conjugated with different natural and unnatural amino acids or decanoic acid (DEC) through an ester bond at one or more available hydroxyl groups. In vitro activity of the modified compounds was determined against Mycobacterium spp. and representative Gram-positive and Gram-negative bacteria. Intracellular activity was evaluated in J774A.1 mouse macrophages infected with Mycobacterium tuberculosis (H37Rv). RESULTS: Acylation of SQ997 and SQ641 with amino undecanoic acid (AUA) improved in vitro activity against most of the bacteria tested. Conjugation of SQ922 with DEC, but not AUA, improved its activity against Gram-positive bacteria. In the presence of efflux pump inhibitor phenylalanine arginine ß-naphthyl amide, MICs of SQ997-AUA, SQ641-AUA and SQ922-DEC compounds improved even further against drug-susceptible and drug-resistant Staphylococcus aureus. In Gram-negative bacteria, EDTA-mediated permeabilization caused 4- to 16-fold enhancement of the activity of AUA-conjugated SQ997, SQ922 and SQ641. Conjugation of all three capuramycin analogues with AUA improved intracellular killing of H37Rv in murine macrophages. CONCLUSIONS: Conjugation of capuramycin analogues with AUA or DEC enhanced in vitro activity, extended the spectrum of activity in Gram-positive bacteria and increased intracellular activity against H37Rv.

Genomic alterations link Rho family of GTPases to the highly invasive phenotype of pancreas cancer.

Pancreas ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically presents as advanced, unresectable disease. This invasive tendency, coupled with intrinsic resistance to standard therapies and genome instability, are major contributors to poor long-term survival. The genetic elements governing the invasive propensity of PDAC have not been well elucidated. Here, in the course of validating resident genes in highly recurrent and focal amplifications in PDAC, we have identified Rio Kinase 3 (RIOK3) as an amplified gene that alters cytoskeletal architecture as well as promotes pancreatic ductal cell migration and invasion. We determined that RIOK3 promotes its invasive activities through activation of the small G protein, Rac. This genomic and functional link to Rac signaling prompted a genome wide survey of other components of the Rho family network, revealing p21 Activated Kinase 4 (PAK4) as another amplified gene in PDAC tumors and cell lines. Like RIOK3, PAK4 promotes pancreas ductal cell motility and invasion. Together, the genomic and functional profiles establish the Rho family GTP-binding proteins as integral to the hallmark invasive nature of this lethal disease.

The differentiation and stress response factor XBP-1 drives multiple myeloma pathogenesis.

Multiple myeloma (MM) evolves from a highly prevalent premalignant condition termed MGUS. The factors underlying the malignant transformation of MGUS are unknown. We report a MGUS/MM phenotype in transgenic mice with Emu-directed expression of the XBP-1 spliced isoform (XBP-1s), a factor governing unfolded protein/ER stress response and plasma-cell development. Emu-XBP-1s elicited elevated serum Ig and skin alterations. With age, Emu-xbp-1s transgenics develop features diagnostic of human MM, including bone lytic lesions and subendothelial Ig deposition. Furthermore, transcriptional profiles of Emu-xbp-1s lymphoid and MM cells show aberrant expression of known human MM dysregulated genes. The similarities of this model with the human disease, coupled with documented frequent XBP-1s overexpression in human MM, serve to implicate XBP-1s dysregulation in MM pathogenesis.

Synergistic interactions of SQ109, a new ethylene diamine, with front-line antitubercular drugs in vitro.

OBJECTIVES: The purpose of this study was to determine interactions of SQ109, a new asymmetric diamine tuberculosis (TB) drug candidate, with existing antitubercular drugs in vitro and assess its potential to improve combination drug activities against Mycobacterium tuberculosis. METHODS: Two-drug combinations at various concentrations below their MICs were tested for growth inhibition of M. tuberculosis using the BACTEC 460 system in vitro. Drug interactions were evaluated based on the quotient values that were derived numerically from the growth indices of cultures treated with a single antibiotic or combination treatment with two antibiotics. RESULTS: SQ109 at 0.5 of its MIC demonstrated strong Synergistic activity with 0.5 MIC isoniazid and as low as 0.1 MIC rifampicin in inhibition of M. tuberculosis growth. Additive effects were observed between SQ109 and streptomycin, but neither synergy nor additive effects were observed with the combination of SQ109 with ethambutol or pyrazinamide. The synergy between SQ109 and rifampicin was also demonstrated using rifampicin-resistant (RIFR) M. tuberculosis strains; SQ109 lowered the MIC of rifampicin for these drug-resistant strains. CONCLUSIONS: SQ109 interacts Synergistically with isoniazid and rifampicin, two of the most important front-line TB drugs. This finding supports efforts to further evaluate new combination therapies containing SQ109 in experimental animal models of TB that emulate future clinical trial studies in humans.

The PTEN and INK4A/ARF tumor suppressors maintain myelolymphoid homeostasis and cooperate to constrain histiocytic sarcoma development in humans.

Histiocytic sarcoma (HS) is a rare malignant proliferation of histiocytes of uncertain molecular pathogenesis. Here, genetic analysis of coincident loss of Pten and Ink4a/Arf tumor suppressors in the mouse revealed a neoplastic phenotype dominated by a premalignant expansion of biphenotypic myelolymphoid cells followed by the development of HS. Pten protein loss occurred only in the histiocytic portion of tumors, suggesting a stepwise genetic inactivation in the generation of HS. Similarly, human HS showed genetic or epigenetic inactivation of PTEN, p16(INK4A), and p14(ARF), supporting the relevance of this genetically engineered mouse model of HS. These genetic and translational observations establish a cooperative role of Pten and Ink4a/Arf in the development of HS and provide mechanistic insights into the pathogenesis of human HS.

High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients.

To identify genetic events underlying the genesis and progression of multiple myeloma (MM), we conducted a high-resolution analysis of recurrent copy number alterations (CNAs) and expression profiles in a collection of MM cell lines and outcome-annotated clinical specimens. Attesting to the molecular heterogeneity of MM, unsupervised classification using nonnegative matrix factorization (NMF) designed for array comparative genomic hybridization (aCGH) analysis uncovered distinct genomic subtypes. Additionally, we defined 87 discrete minimal common regions (MCRs) within recurrent and highly focal CNAs. Further integration with expression data generated a refined list of MM gene candidates residing within these MCRs, thereby providing a genomic framework for dissection of disease pathogenesis, improved clinical management, and initiation of targeted drug discovery for specific MM patients.

915 MHz microwaves and 50 Hz magnetic field affect chromatin conformation and 53BP1 foci in human lymphocytes from hypersensitive and healthy persons.

We used exposure to microwaves from a global system for mobile communication (GSM) mobile phone (915 MHz, specific absorption rate (SAR) 37 mW/kg) and power frequency magnetic field (50 Hz, 15 muT peak value) to investigate the response of lymphocytes from healthy subjects and from persons reporting hypersensitivity to electromagnetic field (EMF). The hypersensitive and healthy donors were matched by gender and age and the data were analyzed blind to treatment condition. The changes in chromatin conformation were measured with the method of anomalous viscosity time dependencies (AVTD). 53BP1 protein, which has been shown to colocalize in foci with DNA double strand breaks (DSBs), was analyzed by immunostaining in situ. Exposure at room temperature to either 915 MHz or 50 Hz resulted in significant condensation of chromatin, shown as AVTD changes, which was similar to the effect of heat shock at 41 degrees C. No significant differences in responses between normal and hypersensitive subjects were detected. Neither 915 MHz nor 50 Hz exposure induced 53BP1 foci. On the contrary, a distinct decrease in background level of 53BP1 signaling was observed upon these exposures as well as after heat shock treatments. This decrease correlated with the AVTD data and may indicate decrease in accessibility of 53BP1 to antibodies because of stress-induced chromatin condensation. Apoptosis was determined by morphological changes and by apoptotic fragmentation of DNA as analyzed by pulsed-field gel electrophoresis (PFGE). No apoptosis was induced by exposure to 50 Hz and 915 MHz microwaves. In conclusion, 50 Hz magnetic field and 915 MHz microwaves under specified conditions of exposure induced comparable responses in lymphocytes from healthy and hypersensitive donors that were similar but not identical to stress response induced by heat shock.

Pharmacodynamics and pharmacokinetics of SQ109, a new diamine-based antitubercular drug.

SQ109 is a novel [1,2]-diamine-based ethambutol (EMB) analog developed from high-throughput combinatorial screening. The present study aimed at characterizing its pharmacodynamics and pharmacokinetics. The antimicrobial activity of SQ109 was confirmed in vitro (Mycobacterium tuberculosis-infected murine macrophages) and in vivo (M. tuberculosis-infected C57BL/6 mice) and compared to isoniazid (INH) and EMB. SQ109 showed potency and efficacy in inhibiting intracellular M. tuberculosis that was similar to INH, but superior to EMB. In vivo oral administration of SQ109 (0.1-25 mg kg(-1) day(-1)) to the mice for 28 days resulted in dose-dependent reductions of mycobacterial load in both spleen and lung comparable to that of EMB administered at 100 mg kg(-1) day(-1), but was less potent than INH at 25 mg kg(-1) day(-1). Monitoring of SQ109 levels in mouse tissues on days 1, 14 and 28 following 28-day oral administration (10 mg kg(-1) day(-1)) revealed that lungs and spleen contained the highest concentration of SQ109, at least 10 times above its MIC. Pharmacokinetic profiles of SQ109 in mice following a single administration showed its C(max) as 1038 (intravenous (i.v.)) and 135 ng ml(-1) (p.o.), with an oral T(max) of 0.31 h. The elimination t(1/2) of SQ109 was 3.5 (i.v.) and 5.2 h (p.o.). The oral bioavailability was 4%. However, SQ109 displayed a large volume of distribution into various tissues. The highest concentration of SQ109 was present in lung (>MIC), which was at least 120-fold (p.o.) and 180-fold (i.v.) higher than that in plasma. The next ranked tissues were spleen and kidney. SQ109 levels in most tissues after a single administration were significantly higher than that in blood. High tissue concentrations of SQ109 persisted for the observation period (10 h). This study demonstrated that SQ109 displays promising in vitro and in vivo antitubercular activity with favorable targeted tissue distribution properties.

Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors.

In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53-HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.

Inhibition of p53 activity in vitro and in living cells by a synthetic peptide derived from its core domain.

Much effort was expended to develop anti-cancer drugs that restore the function of the p53 tumor suppressor protein. However, the p53 activity might be harmful to the organism by amplifying side effects of chemotherapy. Therefore, under certain conditions, inhibition of p53 can serve to prevent inappropriately triggered apoptosis in normal tissues. We have identified a short 22-mer peptide derived from the p53 core domain (peptide 14), which can inhibit p53 specific DNA binding. Upon introduction in living cells, peptide 14 inhibited the ability of p53 to transactivate a reporter gene. Moreover, peptide 14 blocked p53-induced apoptosis in two different cell lines. Peptide 14-mediated inhibition of p53 activity appears to operate via the binding of peptide to the core and/or C-terminal domains of the p53 protein. Our findings provide a basis for the development of a novel approach aimed at the inhibition of p53. This could be essential for the protection from cell death in tissues thus suppressing for example neurodegenerative process or side effects of radio- or chemotherapy.