A novel anti-CD37 antibody-drug conjugate with multiple anti-tumor mechanisms for the treatment of B-cell malignancies.

CD37 has gathered renewed interest as a therapeutic target in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL); however, CD37-directed antibody-drug conjugates (ADCs) have not been explored. Here, we identified a novel anti-CD37 antibody, K7153A, with potent in vitro activity against B-cell lines through multiple mechanisms including apoptosis induction, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. The antibody was conjugated to the maytansinoid, DM1, a potent antimicrotubule agent, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the resulting ADC, IMGN529, retained the intrinsic antibody activities and showed enhanced cytotoxic activity from targeted payload delivery. In lymphoma cell lines, IMGN529 induced G2/M cell cycle arrest after internalization and lysosomal processing to lysine-N(ε)-SMCC-DM1 as the sole intracellular maytansinoid metabolite. IMGN529 was highly active against subcutaneous B-cell tumor xenografts in severe combined immunodeficient mice with comparable or better activity than rituximab, a combination of cyclophosphamide, vincristine, and prednisone, or bendamustine. In human blood cells, CD37 is expressed in B cells at similar levels as CD20, and IMGN529 resulted in potent and specific depletion of normal and CLL B cells. These results support evaluation of the CD37-targeted ADC, IMGN529, in clinical trials in patients with B-cell malignancies including NHL and CLL.

The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids.

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50∼70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈20 µM.

Caged O-phosphorothioyl amino acids as building blocks for Fmoc-based solid phase peptide synthesis.

The synthesis of 1-(2-nitrophenylethyl) caged O-phosphorothioylserine, -threonine and -tyrosine derivatives is reported. These amino acid building blocks can be directly incorporated into peptides by Fmoc-based solid phase synthesis as their pentafluorophenyl esters or as symmetric anhydrides. Upon irradiation with UV light, the thiophosphate group, representing a hydrolysis resistant phosphate analog, is revealed.

Inhibitor of the glutamate vesicular transporter (VGLUT).

The vesicular glutamate transporter (VGLUT) is responsible for the uptake of the excitatory amino acid, L-glutamate, into synaptic vesicles. VGLUT activity is coupled to an electrochemical gradient driven by a vacuolar ATPase and stimulated by low Cl-. VGLUT has relatively low affinity (K(m) = 1-3 mM) for glutamate and is pharmacologically and structurally distinct from the Na+-dependent, excitatory amino acid transporters (EAATs) found on the plasma membrane. Because glutamatergic neurotransmission begins with vesicular release, compounds that block the uptake of glutamate into the vesicle may reduce excitotoxic events. Several classes of competitive VGLUT inhibitors have emerged including amino acids and amino acid analogs, fatty acids, azo dyes, quinolines and alkaloids. The potency with which these agents inhibit VGLUT varies from millimolar (amino acids) to nanomolar (azo dyes) concentrations. These inhibitors represent highly diverse structures and have collectively begun to reveal key pharmacophore elements that may elucidate the key interactions important to binding VGLUT. Using known inhibitor structures and preliminary molecular modeling, a VGLUT pharmacophore is presented that will aid in the design of new, highly potent and selective agents.

Synthesis and in vitro pharmacology of substituted quinoline-2,4-dicarboxylic acids as inhibitors of vesicular glutamate transport.

The vesicular glutamate transport (VGLUT) system selectively mediates the uptake of L-glutamate into synaptic vesicles. Uptake is linked to an H+-ATPase that provides coupling among ATP hydrolysis, an electrochemical proton gradient, and glutamate transport. Substituted quinoline-2,4-dicarboxylic acids (QDCs), prepared by condensation of dimethyl ketoglutaconate (DKG) with substituted anilines and subsequent hydrolysis, were investigated as potential VGLUT inhibitors in synaptic vesicles. A brief panel of substituted QDCs was previously reported (Carrigan et al. Bioorg. Med. Chem. Lett. 1999, 9, 2607-2612)(1) and showed that certain substituents led to more potent competitive inhibitors of VGLUT. Using these compounds as leads, an expanded series of QDC analogues were prepared either by condensation of DKG with novel anilines or via aryl-coupling (Suzuki or Heck) to dimethyl 6-bromoquinolinedicarboxylate. From the panel of almost 50 substituted QDCs tested as inhibitors of the VGLUT system, the 6-PhCH=CH-QDC (K(i) = 167 microM), 6-PhCH2CH2-QDC (K(i) = 143 microM), 6-(4'-phenylstyryl)-QDC (K(i) = 64 microM), and 6-biphenyl-4-yl-QDC (K(i) = 41 microM) were found to be the most potent blockers. A preliminary assessment of the key elements needed for binding to the VGLUT protein based on the structure-activity relationships for the panel of substituted QDCs is discussed herein. The substituted QDCs represent the first synthetically derived VGLUT inhibitors and are promising templates for the development of selective transporter inhibitors.

Lorvotuzumab mertansine, a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models.

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5-10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.

The engineering of membrane-permeable peptides.

Reversible lipid attachment was investigated as a means to deliver small peptides into cells. Two labile straight chain alkyl motifs were developed: a cysteine dodecane disulfide (Cdd) building block and a tyrosine- or serine-myristate ester. Both moieties are cleaved on cell internalization and are compatible with Fmoc solid phase peptide synthesis. A series of fluorophore-labeled peptides that varied in lipophilic content, net charge, and charge distribution were synthesized. The peptides were screened for cellular uptake efficiency as monitored by fluorescence microscopy. Effective peptide transport is based on a distributed net positive charge introduced as lysine residues at the C and/or N terminus of the peptide and the presence of a hydrophobic domain exhibiting an estimated log P4.0. The incorporation of labile lipid motifs into peptides enhances lipophilic character of the peptides and contributes to cellular uptake with minimal alteration to the native sequence.

Zinc is an essential cofactor for type I isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). This is an essential reaction in the mevalonate pathway for biosynthesis of isoprenoid compounds. A crystal structure of Escherichia coli type I IPP isomerase shows a his3glu2 octahedral metal binding site (Durbecq, V. et al. EMBO, 2001, 20, 1530-1537). A metal ion analysis of recombinant E. coli type I IPP isomerase purified from metal-free buffer or buffer containing 10 muM ZnCl2 and 10 muM MnCl2 indicated that the protein contained one atom of Zn2+ per molecule. The metal content and the activity of the enzyme did not change when dialyzed for 6 h against metal-free buffer but rapidly decreased upon dialysis against buffer containing o-phenanthroline. Structural and catalytic roles for Zn2+ are discussed.