Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinasesignaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short-and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.A ntagonizing the function of a protein is an effective strategy for determining its role within a cellular context. For some enzymes and/or receptor proteins, this can be accomplished by incubation with a small molecule inhibitor or antagonist. However, specific small molecule inhibitors have not been discovered or developed for many proteins. As alternative strategies, gene deletion and RNAi approaches function at the DNA and mRNA levels to reduce protein expression, thus offering the ability to control function even for those proteins lacking a specific inhibitor (1, 2). However, such nucleic acid-based approaches do not afford the same rapid, direct assessment of protein function as posttranslational intervention; furthermore, in vivo application of these strategies is even today still complicated and relatively cumbersome. Combining the attractive qualities of both RNAi and small molecule inhibitor approaches, we developed a strategy, proteolysis targeting chimeras (PROTACs) for targeted posttranslational knockdown of proteins. Each PROTAC is heterodimeric, consisting of an E3 ubiquitin ligase-binding moiety linked to a ligand that binds to the target protein (3). As such, each PROTAC recruits i...

The traditional method for in vitro folding of disulfide-containing proteins is slow and involves a redox buffer of glutathione and glutathione disulfide. To increase the folding rate and to gain insight into the folding process, we replaced glutathione, an aliphatic thiol, with a commercially available aromatic thiol, 4-mercaptobenzeneacetate (1). Aromatic thiol 1 was selected due to its enhanced nucleophilicity and its enhanced leaving-group ability relative to glutathione at pH 7.7. To demonstrate the advantages of 1, the folding of reduced and scrambled RNase A at pH 7.0 and 7.7 in the presence of 1 and glutathione was investigated. For each set of folding conditions, the optimum concentration of each thiol was initially determined and then the folding rates in the presence of each thiol were measured concurrently. In all cases examined, the folding rate enhancement with the aromatic thiol was 5- 6-fold. Furthermore, under similar conditions folding rates were almost identical with either reduced or scrambled RNase A. In addition the 5-6-fold folding rate enhancement varied only slightly with pH, 7.0 vs 7.7.

The antileukemic activity (medium effective dose, MED) of a set of 37 carboquinones was modeled using a combination of the electrotopological state (E-state) and molecular connectivity indices with multiple linear regression. A four-variable model gave good statistics: r2 = 0.90, s = 0.21. Using the leave-one-out method, the cross-validation statistics indicate a model useful for prediction: r2press = 0.85, spress = 0.26. The same variables were used to model the optimum effective dose (OD): r2 = 0.88, s = 0.19. The cross-validation statistics indicate a model useful for prediction: r2press = 0.83, spress = 0.23. The descriptor variables are interpreted in terms of the molecular structure.

The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA(+)) ions beginning with tetrabutylammonium (TBA(+)) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA(+)) and tetrahexylammonium (THxA(+)) ions were concentration- and voltage-dependent and occurred only when TAA(+) ions were on the same side as net K(+) efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (K(m)(V(j))) were lower for THxA(+) than TPeA(+), consistent with steric effects within the pore. The K(m)-V(j) relationships for TPeA(+) and THxA(+) were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V(j) field. Bilateral addition of TPeA(+) ions confirmed a common site of interaction within the pore that possessed identical K(m)(V(j)) values for cis-trans concentrations of TPeA(+) ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA(+) block of K(+) currents and bilateral TPeA(+) interactions did not alter V(j)-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA(+) or TPeA(+), indicative of a hydrophobic site within the pore near the site of block.

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.