, and I375A had reduced toxicity to H. virescens. In contrast to our findings with M. sexta, the reduction in toxicity of these mutants was correlated directly with loss of initial binding to H. virescens BBMV, indicating that these residues perform functionally distinct roles in binding and toxicity to different insects. In ligand blots, CryIAb recognized a major 210-kDa peptide in M. sexta BBMV and a 170-kDa peptide in H. virescens BBMV.

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis ␦-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) Ϸ4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering ␦-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Alanine substitutions of loop 3 residues, 438 SGF-SNS 443 , of CryIAb toxin were constructed to study the functional role of these residues in receptor binding and toxicity to Manduca sexta and Heliothis virescens. Experiments with trypsin and insect gut juice enzyme digestions of mutant toxins showed that these mutations did not produce any gross structural changes to the toxin molecule. Bioassay data showed that mutant G439A (alanine substitution of residue Gly 439 ) and F440A significantly reduced toxicity toward M. sexta and H. virescens. In contrast, mutants S438A, S441A, N442A, and S443A were similar or only marginally less toxic (2-3 times) to the insects compared to the wild-type toxin. Binding studies with brush border membrane vesicles prepared from M. sexta and H. virescens midgut membranes revealed that the loss of toxicity of mutants G439A and F440A was attributable to substantially reduced initial binding. Consistent with the initial binding, mutants G349A and F440A showed 3.5 times less binding to M. sexta and H. virescens brush border membrane vesicles, although the off-rate of bound toxins was not affected. The role of hydrophobic residue, Phe 440 , is distinctly different from our previous observation that alanine substitution of Phe 371 at loop 2 of CryIAb did not affect initial binding but reduced irreversible association of the toxin to the receptor or membrane toward M. sexta (Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., and Dean, D. H. (1995) J. Bacteriol. 177, 2276 -2282). Likewise, deletion of relatively hydrophobic CryIAa loop 3 residues, 440 AAGA 443 (D3a), resulted in reduced toxicity to Bombyx mori (>62 times less) and M. sexta (28 times less). The loss of toxicity was correlated with reduced initial binding to midgut vesicles prepared from these insects. However, alanine substitution of residues 437 LSQ 439 (A3a), contiguous to loop 3, altered neither toxicity nor receptor binding toward B. mori or M. sexta. These results suggest that the loop 3 residues of CryIAb and CryIAa toxins establish hydrophobic interactions with the receptor molecule, and mutations at these hydrophobic residues affect initial binding.

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120-and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.

Tubulin is the proposed target for drugs against cancer and helminths and is also a validated target in kinetoplastid parasites. With the aim of identifying new lead compounds against Leishmania sp., tubulin isolated from L. tarentolae was used to screen a 10 000 compound library. One compound, Chembridge No. 7992831 (5), displayed an IC50 of 13 μm against Leishmania tubulin in an in vitro assembly assay and showed a greater than threefold selectivity over mammalian tubulin. Another compound, Chembridge No. 9067250 (8), exhibited good activity against mammalian tubulin (IC50 = 5.0 μm). This compound was also toxic to several cancer cell lines with IC50 values in the region of 1 μM. Subsequent testing of analogues of 8 contained within the library identified two compounds with greater potency against mammalian tubulin (IC50 values of 1.1 and 2.8 μM). The more potent antitubulin agent also showed promising activity against cancer cell lines in vitro, with IC50 values ranging from 0.18 to 0.73 μM.

We conclude that myoglobinuria with myoglobin cast formation can occur following rapamycin administration, and may be a causative factor in the development of unexpected severe acute renal dysfunction.

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Purpose: ABT-888 is an oral inhibitor of PARP 1 and 2 and potentiates activity of platinums in preclinical models. We are conducting a phase I study of ABT-888 on 2 different schedules (7 and 14 day) plus q 3 week carboplatin (C) to identify the recommended phase II dose schedules (RPTD-7and 14 day) for this regimen in patients (pts) with MBC who are: (1) triple negative (TN); or (2) estrogen receptor positive (ER+) with defective Fanconi Anemia (FA) pathway (no FANCD2 foci in tumor). Study design: Eligible pts received C-AUC 5 Q 3weeks (except dose level 1-AUC 6) plus escalating doses of ABT-888 BID on a 7 or 14-day schedule using a 3+3 dose escalation design. Blood samples were collected during cycles 1 and 2 for PAR assay and CTCs isolated to measure gamma H2Ax. We performed 3′-[F-18] Fluoro-3′-deoxythymidine(FLT) PET scans to assess cellular tumor proliferation at baseline, cycle 1 day 7 and 14 and after cycle 3. FDG-PET-CT scans were used to assess response. Results: 22 pts (20-TN, 2-ER+ w/FA defect) with median age of 56.5 yrs (range 31-69) were enrolled on 5 dose levels. Dose level 1 with C at AUC 6 was too toxic with 3 DLTs (Table 1). Further dose escalations were performed with C at AUC-5. Dose reductions in C were made in subsequent cycles for thrombocytopenia, anemia and fatigue. Maximum reduction in thymidine uptake in tumors was seen on day 7 FLT scans. CTCs were isolated using an immunomagnetic negative depletion method on 8 pt samples to date (median-59, range 0-684 CTCs). Gamma H2Ax analysis on sequential samples is ongoing. Four pts have had an unconfirmed partial response (PR) with > 50% tumor shrinkage; 2 of the 4 pts had a defective FA pathway. Conclusions: Thrombocytopenia is the major DLT when ABT-888 is given in combination with C, where lower AUC of C is better tolerated and shows promising activity of this combination. FA deficiency (5/14=27%) seen in this group is consistent with our previous reports. The study is supported U01 CA076576. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5586. doi:1538-7445.AM2012-5586