The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules.

Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with 32 P-labeled DNA quantify Kd values from 10-12 to 10-8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio koff /kon determines Kd . However, for lactose repressor protein (LacI) and an engineered repressor protein ("LLhF") binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below Kd (equilibrium binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10-9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10-8 M should reliably be in the equilibrium binding regime. We also observed effects from multi-valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein-DNA binding and to assess the affinity of fructose-1-kinase for the DNA-LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.

Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over-looked physiological factors.

To bridge biological and biochemical disciplines, the relationship between in vitro protein biochemical function and in vivo activity must be established. Such studies can (a) help determine whether properties measured in simple, dilute solutions extrapolate to the complex in vivo conditions and (b) illuminate cryptic biological factors that are new avenues for study. We have explored the in vivo-in vitro relationship for chimeras built from LacI/GalR transcription regulators. In prior studies of individual chimeras, amino acid changes that altered in vitro DNA binding affinity exhibited correlated changes in in vivo transcription repression. However, discrepancies arose when the two datasets were compared to each other: Although their DNA binding domains were identical and their in vitro binding affinities spanned the same range, their in vivo repression ranges differed by >50-fold. Here, we determined that the presence of endogenous ligand for one chimera further exacerbated the offset, but that different abilities to simultaneously bind and "loop" two DNA operators resolves the discrepancy. Indeed, results suggest that the lac operon can be looped by even weakly interacting repressor dimers. For looping-competent repressors, we measured in vitro binding to the secondary operator. Surprisingly, this was largely insensitive to amino acid changes in the repressor protein, which reflects altered specificity; this supports the emerging view that the locations of specificity determining positions can be unique to each protein homolog. In aggregate, this work illustrates how a comparative approach can enrich understanding of the in vivo-in vitro relationship and suggest unexpected avenues for future study.

Efforts in redesigning the antileukemic drug 6-thiopurine: decreasing toxic side effects while maintaining efficacy.

6-Thiopurine (6TP) is a currently prescribed drug in the treatment of diseases ranging from Crohn's disease to acute lymphocytic leukemia. While its potent mode of action is through incorporation into DNA as a thiol mimic of deoxyguanosine, severe toxicities are associated with its administration which hinder the potential therapeutic application. We have previously reported in vitro that the oxidative metabolites of 6TP, specifically 6-thiouric acid (6TU, K i 7 µM), are potent inhibitors of UDP-glucose dehydrogenase (UDPGDH), an enzyme that is responsible for the formation of UDP-glucuronic acid (UDPGA), an essential substrate that is used in detoxification processes in the liver. An in vivo investigation was undertaken to probe if 6TU inhibits UDPGDH in rat hepatocytes, and it was observed that 6TU does greatly suppress the conjugation of bilirubin with UDPGA. The failed excretion of bilirubin is linked to a majority of the reported toxicities associated with 6TP administration. Efforts were undertaken for the construction of 6TP analogs, substituted at the C8 position, to reduce inhibition of UDPGDH while retaining therapeutic efficacy. Three new 6TP analogs bearing a halogen (Br, Cl, and F) at the C8 position have been achieved over five-synthetic steps in overall yields of 16 to 32%. Each of these analogs were shown to have reduced inhibition towards UDPGDH, with K i values of 192, 163, 215 µM, respectively. In addition, the bromine, chlorine, and fluorine analogs were shown to possess cytotoxicity towards the REH cell line (acute lymphocytic leukemia) having IC50 values of 9.54 µM (±0.97), 3.95 µM (±1.94), and 4.71 µM (±1.40), respectively. These three new 6TP analogs represent the first steps in the redesign of this potent anticancer agent into a better drug that possesses reduced toxic side effects while retaining therapeutic potency.

Inhibition of UDP-glucose dehydrogenase by 6-thiopurine and its oxidative metabolites: Possible mechanism for its interaction within the bilirubin excretion pathway and 6TP associated liver toxicity.

6-Thiopurine (6TP) is an actively prescribed drug in the treatment of various diseases ranging from Crohn's disease and other inflammatory diseases to acute lymphocytic leukemia and non-Hodgkin's leukemia. While 6TP has beneficial therapeutic uses, severe toxicities are also reported with its use, such as jaundice and liver toxicity. While numerous investigations into the mode in which toxicity originates has been undertaken. None have investigated the effects of inhibition towards UDP-Glucose Dehydrogenase (UDPGDH), an oxidative enzyme responsible for UDP-glucuronic acid (UDPGA) formation or UDP-Glucuronosyl transferase (UGT1A1), which is responsible for the conjugation of bilirubin with UDPGA for excretion. Failure to excrete bilirubin leads to jaundice and liver toxicity. We proposed that either 6TP or its primary oxidative excretion metabolites inhibit one or both of these enzymes, resulting in the observed toxicity from 6TP administration. Inhibition analysis of these purines revealed that 6-thiopurine has weak to no inhibition towards UDPGDH with a Ki of 288 µM with regard to varying UDP-glucose, but 6-thiouric (primary end metabolite, fully oxidized at carbon 2 and 8, and highly retained by the body) has a near six-fold increased inhibition towards UDPGDH with a Ki of 7 µM. Inhibition was also observed by 6-thioxanthine (oxidized at carbon 2) and 8-OH-6TP with Ki values of 54 and 14 µM, respectively. Neither 6-thiopurine or its excretion metabolites were shown to inhibit UGT1A1. Our results show that the C2 and C8 positions of 6TP are pivotal in said inhibition towards UDPGDH and have no effect upon UGT1A1, and that blocking C8 could lead to new analogs with reduced, if not eliminated jaundice and liver toxicities.