Over the past decade, therapeutics that target subsets of the 518 human protein kinases have played a vital role in the fight against cancer. Protein kinases are typically targeted at the adenosine triphosphate (ATP) binding cleft by type I and II inhibitors, however, the high sequence and structural homology shared by protein kinases, especially at the ATP binding site, inherently leads to polypharmacology. In order to discover or design truly selective protein kinase inhibitors as both pharmacological reagents and safer therapeutic leads, new efforts are needed to target kinases outside the ATP cleft. Recent advances include the serendipitous discovery of type III inhibitors that bind a site proximal to the ATP pocket as well as the truly allosteric type IV inhibitors that target protein kinases distal to the substrate binding pocket. These new classes of inhibitors are often selective but usually display moderate affinities. In this review we will discuss the different classes of inhibitors with an emphasis on bisubstrate and bivalent inhibitors (type V) that combine different inhibitor classes. These inhibitors have the potential to couple the high affinity and potency of traditional active site targeted small molecule inhibitors with the selectivity of inhibitors that target the protein kinase surface outside ATP cleft.
Proton transfer reactions are ubiquitous in enzymes and utilize active site residues as general acids and bases. Crystal structures and site-directed mutagenesis are routinely used to identify these residues, but assessment of their catalytic contribution remains a major challenge. In principle, effective molarity measurements, in which exogenous acids/bases rescue the reaction in mutants lacking these residues, can estimate these catalytic contributions. However, these exogenous moieties can be restricted in reactivity by steric hindrance or enhanced by binding interactions with nearby residues, thereby resulting in over- or underestimation of the catalytic contribution, respectively. With these challenges in mind, we investigated the catalytic contribution of an aspartate general base in ketosteroid isomerase (KSI) by exogenous rescue. In addition to removing the general base, we systematically mutated nearby residues and probed each mutant with a series of carboxylate bases of similar pKa but varying size. Our results underscore the need for extensive and multifaceted variation to assess and minimize steric and positioning effects and determine effective molarities that estimate catalytic contributions. We obtained consensus effective molarities of ∼5 × 10(4) M for KSI from Comamonas testosteroni (tKSI) and ∼10(3) M for KSI from Pseudomonas putida (pKSI). An X-ray crystal structure of a tKSI general base mutant showed no additional structural rearrangements, and double mutant cycles revealed similar contributions from an oxyanion hole mutation in the wild-type and base-rescued reactions, providing no indication of mutational effects extending beyond the general base site. Thus, the high effective molarities suggest a large catalytic contribution associated with the general base. A significant portion of this effect presumably arises from positioning of the base, but its large magnitude suggests the involvement of additional catalytic mechanisms as well.
*Fried et al. (Reports, 19 December 2014, p. 1510) demonstrated a strong correlation between reaction rate and the carbonyl stretching frequency of a product analog bound to ketosteroid isomerase oxyanion hole mutants and concluded that the active-site electric field provides 70% of catalysis. Alternative comparisons suggest a smaller contribution, relative to the corresponding solution reaction, and highlight the importance of atomiclevel descriptions. We were excited to see the data of Fried et al.(1) demonstrating a strong correlation between reaction rate and the stretching frequency of the carbonyl group of a product analog bound to a series of ketosteroid isomerase (KSI) oxyanion hole mutants. These data were interpreted in terms of a model, calibrated using vibrational data and molecular dynamics simulations in a series of solvents, which led to the conclusion that the active-site electric field generated by the oxyanion hole and surrounding groups accounts for 10 5 -fold rate enhancement and 70% of the observed catalysis. Based on these findings, it was suggested that electrostatic forces are the dominant contributor to catalysis.Below, we note that the conclusion of a dominant contribution to KSI catalysis relies on comparison to a hypothetical enzyme that provides zero electric field at the position of the carbonyl group and would not hold for a comparison to the corresponding reaction in aqueous solution. The accompanying analysis leading to an estimate of the rate advantage from positioning of KSI's general base is similarly affected. Finally, we note that electrostatic stabilization requires and is linked to positioning of the groups responsible for that stabilization.Conservative mutations, such as the Tyr 16 Phe mutation in the construct employed by Fried et al., often transmute a polar group to a nonpolar group and generate an apolar or hydrophobic environment that is less favorable toward charge accumulation than the polar environment in aqueous solution, and are inhibitory as a result (2). Thus, a "conservative" mutation of the dominant Kraut et al. (2) and correspond to the substrate 5(10)-estren-3,17-dione, because a chemical step is rate-limiting for reaction of this substrate (9). Similar rate enhancements are provided for reactions with the substrate 5-androstene-3,17-dione referred to by Fried et al., although a nonchemical step is partially rate limiting for this substrate (9, 10).
Antibody–drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs.
Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations up to 109-fold relative to the same reaction in aqueous solution. Nevertheless, several other systems, such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7,000-fold better than wild-type KSI, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, carries out the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, non-specific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design is able to fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.
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