Thomas D. Meek

Drug–target residence time and its implications for lead optimization

Much of drug discovery today is predicated on the concept of selective targeting of particular bioactive macromolecules by low-molecular-mass drugs. The binding of drugs to their macromolecular targets is therefore seen as paramount for pharmacological activity. In vitro assessment of drug–target interactions is classically quantified in terms of binding parameters such as IC50 or Kd. This article presents an alternative perspective on drug optimization in terms of drug–target binary complex residence time, as quantified by the dissociative half-life of the drug–target binary complex. We describe the potential advantages of long residence time in terms of duration of pharmacological effect and target selectivity.

Chromophoric peptide substrates for the spectrophotometric assay of HIV-1 protease.

Purified HIV-1 protease hydrolyzes H-Ser-Gln-Asn-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (Peptide 1) and acetyl-Arg-Lys-Ile-Leu-Phe(NO2)-Leu-Asp-Gly-NH2 (Peptide 2) between the (p-nitro)phenylalanyl and leucyl residues. The cleavage of Peptides 1 and 2 resulted in a decrease in uv absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of Peptides 1 and 2 was characterized by a linear time course at substrate turnover of less than 20%. The solubilities of these substrates at pH 5.0 were sufficient to provide initial rate measurements over a concentration range of 0.05-0.5 mM. Steady-state kinetic data and inhibition constants using both spectrophotometric and high performance liquid chromatography (HPLC) analysis of the peptidolysis of these substrates resulted in comparable values.

SYNTHESIS AND ANTIVIRAL ACTIVITY OF A NOVEL CLASS OF HIV-1 PROTEASE INHIBITORS CONTAINING A HETEROCYCLIC P1'-P2' AMIDE BOND ISOSTERE

A novel series of hydroxyethylene-based peptidomimetics that contain 2-substituted nitrogen heterocycles as P1′-P2′ amide bond isosteres has been prepared and evaluated as inhibitors of HIV-1 protease and in vitro HIV-1 replication. Many of these compounds exhibit inhibition constants in the low to subnanomolar range. Structure-activity relationships are discussed.

A radiometric assay for HIV-1 protease

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

Chemical Mechanism of a Cysteine Protease, Cathepsin C, As Revealed by Integration of both Steady-State and Pre-Steady-State Solvent Kinetic Isotope Effects

Cathepsin C, or dipeptidyl peptidase I, is a lysosomal cysteine protease of the papain family that catalyzes the sequential removal of dipeptides from the free N-termini of proteins and peptides. Using the dipeptide substrate Ser-Tyr-AMC, cathepsin C was characterized in both steady-state and pre-steady-state kinetic modes. The pH(D) rate profiles for both log k cat/ K m and log k cat conformed to bell-shaped curves for which an inverse solvent kinetic isotope effect (sKIE) of 0.71 +/- 0.14 for (D)( k cat/ K a) and a normal sKIE of 2.76 +/- 0.03 for (D) k cat were obtained. Pre-steady-state kinetics exhibited a single-exponential burst of AMC formation in which the maximal acylation rate ( k ac = 397 +/- 5 s (-1)) was found to be nearly 30-fold greater than the rate-limiting deacylation rate ( k dac = 13.95 +/- 0.013 s (-1)) and turnover number ( k cat = 13.92 +/- 0.001 s (-1)). Analysis of pre-steady-state burst kinetics in D 2O allowed abstraction of a normal sKIE for the acylation half-reaction that was not observed in steady-state kinetics. Since normal sKIEs were obtained for all measurable acylation steps in the presteady state [ (D) k ac = 1.31 +/- 0.04, and the transient kinetic isotope effect at time zero (tKIE (0)) = 2.3 +/- 0.2], the kinetic step(s) contributing to the inverse sKIE of (D)( k cat/ K a) must occur more rapidly than the experimental time frame of the transient kinetics. Results are consistent with a chemical mechanism in which acylation occurs via a two-step process: the thiolate form of Cys-234, which is enriched in D 2O and gives rise to the inverse value of (D)( k cat/ K a), attacks the substrate to form a tetrahedral intermediate that proceeds to form an acyl-enzyme intermediate during a proton transfer step expressing a normal sKIE. The subsequent deacylation half-reaction is rate-limiting, with proton transfers exhibiting normal sKIEs. Through derivation of 12 equations describing all kinetic parameters and sKIEs for the proposed cathepsin C mechanism, integration of both steady-state and pre-steady-state kinetics with sKIEs allowed the provision of at least one self-consistent set of values for all 13 rate constants in this cysteine proteases chemical mechanism. Simulation of the resulting kinetic profile showed that at steady state approximately 80% of the enzyme exists in an active-site cysteine-acylated form in the mechanistic pathway. The chemical and kinetic details deduced from this work provide a potential roadmap to help steer drug discovery efforts for this and other disease-relevant cysteine proteases.

USE OF STEADY STATE KINETIC METHODS TO ELUCIDATE THE KINETIC AND CHEMICAL MECHANISMS OF RETROVIRAL PROTEASES

Despite the current plethora of structural data of HIV-1 protease and the availability of potent inhibitors, whose structures are based in part on the presumed mechanism of action of this enzyme, our actual understanding of its chemical mechanism has been until now based largely on the precedents of the mammalian and fungal aspartic proteases and static three-dimensional data. The available steady state kinetic data of the protease, as reviewed here, constitute a first step in a detailed description of the mechanism of the enzyme to complement the structural data.

Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36

Significance Epigenetic control by methylation of histones is essential in development. Loss of regulation of methylation pathways is involved in developmental disorders and oncogenesis. Despite interest in NSD2, there have been no selective inhibitors reported. Analogs designed to mimic the NSD2 transition state structure are potential enzyme inhibitors. A combination of experimental kinetic isotope effects and quantum chemistry was used to define the subangstrom details of reaction chemistry at the transition state of NSD2. Electrostatic potential maps of reactants and transition states provide a high-resolution map of reaction chemistry and a blueprint for design of transition state analogs for this mechanism of epigenetic regulation. Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf–Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-14C and 36S and secondary Me-3H3, Me-2H3, 5′-14C, and 5′-3H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

Kinetic Mechanism and Rate-Limiting Steps of Focal Adhesion Kinase-1

Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH(2), FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k(cat) (0.052 +/- 0.001 s(-1)), K(MgATP) (1.2 +/- 0.1 microM), K(iMgATP) (1.3 +/- 0.2 microM), K(FAK-tide) (5.6 +/- 0.4 microM), and K(iFAK-tide) (6.1 +/- 1.1 microM) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the betagamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-(18)O(4)]ATP in the presence of 1 mM [gamma-(18)O(4)]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k(x)/k(cat) = 0.14 +/- 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k(cat)/K(FAK-tide), while k(cat) and k(cat)/K(MgATP) were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k(cat)/K(m) for MgATP but not on k(cat)/K(m) for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (k(cat)) is rate-limited by both reversible phosphoryl group transfer (k(forward) approximately 0.2 s(-1) and k(reverse) approximately 0.04 s(-1)) and a slow step (k(conf) approximately 0.1 s(-1)) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.

HIV‐1 Protease as a Potential Target for Anti‐AIDS Therapy

Human immunodeficiency virus (HIV), the causative agent of the acquired immune deficiency syndrome (AIDS), is a member of the family of retroviruses, Retroviridiae. I As the genome of the retroviruses consists of RNA, the hallmark of retroviral infection of a host cell is the conversion of this RNA genome to DNA by virus-mediated reverse transcription, followed by the stable integration of the retroviral DNA genome into the cell’s chromosomes.2 From this point the expression of the integrated retroviral genes, known as the provirus, by the replicative machinery of the host cell, produces new retroviral particles that effect either cytopathogenesis or cellular transformation and advance infection to other cells. Following the installment of the provirus into the host cell genome, the proviral genes of HIV and other retroviruses are expressed, and their products are assembled into new virion particles. The RNA transcripts of the provirus are synthesized by the cellular RNA polymerase 11, which then undergo posttranscriptional processing. These primary transcripts serve two functions: as the source of mRNA from which viral proteins are made and as the genomic RNA that is ultimately packaged into new virions. Translation of the proviral mRNA results in the synthesis of viral structural proteins and enzymes, many of which exist as precursors within polyp rote in^.^ Posttranslational modifications of the proviral gene products are performed by cellular enzymes to prepare the viral proteins for proper packaging within new virions. These processes include myri~toylation~ of the N-termini of the viral polyproteins and glycosylation of the envelope proteins. Virion assembly is initiated within the cell membrane when “immature” particles, composed of a glycoprotein envelope, genomic RNA, and viral polyproteins, begin to form and bud from the cell. Maturation of fully formed immature virion particles that have budded from the cell is effected by the action of a virally encoded enzyme, the retroviral p r ~ t e a s e . ~ * ~ ~ The protease specifically cleaves the encapsulated viral polyproteins into the functional enzymes and structural proteins of the virion core. The resulting mature virion particles are now able to promote a new infection in an adjacent T lymphocyte. In retroviruses, the proteins that ultimately make up the virion core and the enzymes essential to viral replication are the respective products of the gag and pol genes (FIG. 1). Direct translation of gag results in a 55 kilodalton (kDa) polyprotein, Pr55g“g. This precursor contains the precursor forms of the structural proteins of the virion core in a single polypeptide chain, arranged as H 2 N p 17-p24-p1 -p9(p7)-p6-COOH (FIG. 1). ‘CL l5 The eventual proteolytic processing of Pr55g‘g generates the gag proteins, each of which has a specific role in the fully formed virion: the matrix protein (p17, MA;16) constitutes the membrane-associated outer shell of the virion capsid; the rod-shaped

Nonpeptide HIV protease inhibitors designed to replace a bound water

Abstract Cyclic nonpeptide molecules were designed and synthesized with the goal of displacing the conserved flap-associated water of HIV-1 protease. Several such molecules were competitive inhibitors with micromolar inhibition constants, and their structure-activity relationships were consistent with the design hypothesis.