Intact Protein Mass Spectrometry of Cell Culture Harvest Guides Cell Line Development for Trispecific Antibodies.

IgG-like multispecific antibodies with asymmetric constructs have become widely used formats for therapeutic applications in recent years. Correct assembly of the subunits in this class of therapeutics is a critical quality attribute (CQA) with direct impact on biological activity. Therefore, early drug development efforts such as clone selection during cell line development must be guided by information on potential chain mispairing to enable timely decision making and risk mitigation. Here we describe a high-throughput analytical platform based on denaturing size-exclusion ultraperformance liquid chromatography (UPLC) coupled with intact protein mass spectrometry for profiling of mispairing and other product-related impurities, including half antibodies. This method can be performed directly on the clarified cell culture harvest fluid without the need for Protein A purification or other sample preparations and provides unbiased information on the product quality of the clones and the effect of growth conditions in a fast and cost-effective manner. Screening large numbers of clones expressing different trispecific antibody (tsAb) constructs revealed that although chain mispairing primarily depends on the antibody sequence and structure, it is also a characteristic of the clone. In addition, different growth conditions may affect the type and distribution of half antibodies and mispaired species impurities but not the quality ranking of the clones.

Identification of triazinoindol-benzimidazolones as nanomolar inhibitors of the Mycobacterium tuberculosis enzyme TDP-6-deoxy-d-xylo-4-hexopyranosid-4-ulose 3,5-epimerase (RmlC).

High-throughput screening of 201,368 compounds revealed that 1-(3-(5-ethyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one (SID 7975595) inhibited RmlC a TB cell wall biosynthetic enzyme. SID 7975595 acts as a competitive inhibitor of the enzyme's substrate and inhibits RmlC as a fast-on rate, fully reversible inhibitor. An analog of SID 7975595 had a K(i) of 62nM. Computer modeling showed that the binding of the tethered two-ringed system into the active site occurred at the thymidine binding region for one ring system and the sugar region for the other ring system.

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A.

The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3',5'-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K(I)=43.4+/-7.2muM and k(cart)=0.007+/-0.0006 min(-1)). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K(d)=72 microM)>Sp-cGMPS (124), Sp-cAMPS (182)>GMP (1517), Rp-cAMPS (3762), AMP (4370 microM). NAD(+), neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.

Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase.

Adenylosuccinate lyase (ASL) of Bacillus subtilis contains three conserved histidines, His(68), His(89), and His(141), identified by affinity labeling and site-directed mutagenesis as critical to the intersubunit catalytic site. The pH-V(max) profile for wild-type ASL is bell-shaped (pK (1) = 6.74 and pK (2) = 8.28). Only the alkaline side changes with temperature, characteristic of histidine pKs. To identify determinants of pK (2) in the enzyme-substrate complex, we replaced residues at two positions close to His(68) (but not to His(89) or His(141)) in the structure. Compared with the specific activity of 1.75 mumol adenylosuccinate reacting/min/mg of wild-type enzyme at pH 7.0, mutant enzymes D69E, D69N, R310Q, and R310K exhibit specific activities of 0.40, 0.04, 0.00083, and 0.10, respectively. While D69E has a K (m) for adenylosuccinate similar to that of wild-type ASL, D69N and R310K exhibit modest increases in K (m), and R310Q has an 11-fold increase in K (m). The mutant enzymes show no significant change in molecular weight or secondary structure. The major change is in the pH-V(max) profile: pK (2) is 8.48 for the D69E mutant and is decreased to 7.83 in D69N, suggesting a proximal negative charge is needed to maintain the high pK of 8.28 observed for wild-type enzyme and attributed to His(68). Similarly, R310Q exhibits a decrease in its pK (2) (7.33), whereas R310K shows little change in pK (2) (8.24). These results suggest that Asp(69) interacts with His(68), that Arg(310) interacts with and orients the beta-carboxylate of Asp(69), and that His(68) must be protonated for ASL to be active.

A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.

Types 1 and 2 diabetes affect some 380 million people worldwide. Both ultimately result from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with a peak percentage (∼2%) engaged in the cell cycle in the first year of life. In embryonic life and after early childhood, beta cell replication is barely detectable. Whereas beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts. Hence, there remains an urgent need for antidiabetic therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, using a high-throughput small-molecule screen (HTS), we find that analogs of the small molecule harmine function as a new class of human beta cell mitogenic compounds. We also define dual-specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine and the nuclear factors of activated T cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation and differentiation. Using three different mouse and human islet in vivo-based models, we show that harmine is able to induce beta cell proliferation, increase islet mass and improve glycemic control. These observations suggest that harmine analogs may have unique therapeutic promise for human diabetes therapy. Enhancing the potency and beta cell specificity of these compounds are important future challenges.

A protein kinase C inhibitor induces phenotypic reversion of ras-transformed pancreatic cancer cells and cooperatively blocks tumor cell proliferation with an anti- ras peptide.

PURPOSE: We have previously found that the staurosporine derivative, CGP 41 251, that has a high specificity for inhibiting protein kinase C (PKC), selectively blocks oncogenic ras-p21-induced oocyte maturation and that PKC and jun-N-terminal kinase (JNK), with which oncogenic ras-p21 directly interacts, reciprocally require each other's activation. We sought to determine whether CGP 41 251 blocks proliferation of ras-transformed mammalian cells and whether it synergistically exerts this effect with a ras-p21 peptide (residues 96-110) that interferes with the interaction of ras-p21 with JNK. METHODS: We incubated ras-transformed rat pancreatic cancer TUC-3 cells and their normal counterpart pancreatic acinar BMRPA1 cells with CGP 42 251 alone and in the presence of the ras-p21 96-110 peptide, both in pre- and post-monolayer phases and determined cell counts and morphology and, for TUC-3 cells, their ability to grow on soft agar. In the post-monolayer experiments, we also evaluated these parameters after withdrawal of these agents. RESULTS: CGP 41 251, but not its inactive analogue, CGP 42 700, blocked pre-monolayer growth and reduced post-monolayer cell counts of both TUC-3 and BMRPA1 cells (IC(50) 0.28 and 0.35 micro M, respectively). After 2 weeks of treatment, all the remaining TUC-3 cells exhibited the untransformed phenotype. Withdrawal of CGP 41 251 resulted in almost complete regrowth of the normal BMRPA1 cells while the reverted TUC-3 cells grew much more slowly. These effects were greatly enhanced by the presence of the ras-p21 96-110 peptide. CONCLUSIONS: CGP 41 251 strongly blocks growth of ras-transformed pancreatic cancer cells by causing cell death and by induction of phenotypic reversion. The enhancement of this effect by the ras-p21 96-110 peptide indicated synergy between it and CGP 41 251, allowing it to block proliferation of the transformed cells selectively. These findings suggest the possibility of using these two agents in anticancer therapy.

Miniaturized High-Throughput Fluorescent Assay for Conversion of NAD(P)H to NAD(P).

A miniaturized fluorescent assay is described that monitors the conversion of NADPH to NADP+. The same assay format may also be used to measure NADH to NAD+ conversion. Examples of assay development and validation results are presented to illustrate the use of this protocol to screen an enzyme that consumes NADPH as a cofactor during conversion of substrate to a reduced product. Enzymatic assays are carried out low volume 384-well plates, in which the turnover of NADPH is monitored by the decrease in fluorescent emission at 460 nm between an initial fluorescence measurement and a second reading after 90 minutes. A follow-up assay is used to rule out false positive artifacts arising from compounds that fluoresce at 460 nm.

Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens.

Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K(I) approximately 0.1 microM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.

Two novel mutant human adenylosuccinate lyases (ASLs) associated with autism and characterization of the equivalent mutant Bacillus subtilis ASL.

An Australian patient with autism was found to be heterozygous for two mutations in the gene encoding adenylosuccinate lyase (ASL), resulting in the protein mutations E80D and D87E. The patient's mother carried only the E80D mutation. The equivalent positions are 62 and 69 in Bacillus subtilis ASL. Although both human and B. subtilis enzymes normally have Asp at position 87 (or 69), the B. subtilis ASL has Ile and Asp at 62 and 65, respectively, whereas human ASL has Glu and Arg at the equivalent positions. We have constructed, expressed, and purified the double mutant I62E/D65R as a "humanized" normal B. subtilis enzyme to compare with enzymes with a single mutation at position 62 (I62D/D65R), at position 69 (I62E/D65R/D69E), or at both positions (I62D/D65R/D69E). V(max) for conversion of adenylosuccinate to AMP and fumarate is 0.57 micromol/min/mg for I62E/D65R, 0.064 micromol/min/mg for I62D/D65R, 0.27 micromol/min/mg for I62E/D65R/D69E, and 0.069 micromol/min/mg for I62D/D65R/D69E. The K(m) for adenylosuccinate is elevated in the X62D mutants, and I62D/D65R is the least stable of these ASLs at 37 degrees C. The CD spectra of mutant and wild type enzymes are similar; thus, there are no appreciable structural changes. Clearly the Asp(62) causes the most drastic effect on ASL function, whereas the Glu(69) mutation produces only modest change. These results emphasize the importance of expanding tests for ASL deficiency to individuals with developmental delay of any severity, including individuals with autistic spectrum disorder. This study further demonstrates the usefulness of the B. subtilis ASL as a model to mimic the defective enzyme in ASL deficiency.