Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer's disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

The DNA Polymerase Gamma R953C Mutant Is Associated with Antiretroviral Therapy-Induced Mitochondrial Toxicity.

We found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-γ) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-γ mutant binding affinity for dCTP is 8-fold less than that of the wild type. The R953C mutant shows a 4-fold decrease in discrimination of analog nucleotides relative to the wild type. R953 is located on the "O-helix" that forms the substrate deoxynucleoside triphosphate (dNTP) binding site; the interactions of R953 with E1056 and Y986 may stabilize the O-helix and affect polymerase activity.

Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol.

Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase γ (mtDNA Pol γ) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPol-mediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol γ that may manifest in toxicity.

Potent Inhibitors Active against HIV Reverse Transcriptase with K101P, a Mutation Conferring Rilpivirine Resistance.

Catechol diether compounds have nanomolar antiviral and enzymatic activity against HIV with reverse transcriptase (RT) variants containing K101P, a mutation that confers high-level resistance to FDA-approved non-nucleoside inhibitors efavirenz and rilpivirine. Kinetic data suggests that RT (K101P) variants are as catalytically fit as wild-type and thus can potentially increase in the viral population as more antiviral regimens include efavirenz or rilpivirine. Comparison of wild-type structures and a new crystal structure of RT (K101P) in complex with a leading compound confirms that the K101P mutation is not a liability for the catechol diethers while suggesting that key interactions are lost with efavirenz and rilpivirine.

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ.

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3'-dideoxy-5-fluoro-3'-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3'-dideoxy-3'-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.

A mechanistic and structural investigation of modified derivatives of the diaryltriazine class of NNRTIs targeting HIV-1 reverse transcriptase.

BACKGROUND: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are vital in treating HIV-1 infection by inhibiting reverse transcriptase (RT). Drug toxicity and resistance drive the need for effective new inhibitors with improved physiochemical properties and potent antiviral activity. Computer-aided and structure-based drug design have guided the addition of solubilizing substituents to the diaryltriazine scaffold. These derivatives have markedly improved solubility and maintain low nanomolar antiviral activity against RT. The molecular and structural basis of inhibition for this series was determined to facilitate future inhibitor development with improved pharmacological profiles. METHODS: The molecular mechanism of inhibition was investigated using transient-state kinetic analysis. Crystal structures of RT in complex with each inhibitor were obtained to investigate the structural basis of inhibition. RESULTS: The diaryltriazine and its morpholine derivative have RT inhibition constants of 9±2nM and 14±4nM, respectively. They adopt differential binding modes within the non-nucleoside inhibitor binding pocket to distort the catalytic site geometry and primer grip regions. The novel morpholinopropoxy substituent extends into the RT/solvent interface of the NNIBP. CONCLUSIONS: Kinetic and structural analyses show that these inhibitors behave as conventional NNRTIs and inhibit the polymerization step. This study confirms that appending solubilizing substituents on the azine ring of diaryltriazine class of NNRTIs that extend into the RT/solvent interface effectively maintains low nanomolar potency and improves physiochemical properties. GENERAL SIGNIFICANCE: The modification of NNRTI scaffolds with solubilizing substituents, which extend into the RT/solvent interface, yields potent antivirals and is an effective strategy for developing novel inhibitors with improved pharmacological properties.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics.

The novel antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3'-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3'-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed.

Crystal structures of HIV-1 reverse transcriptase with picomolar inhibitors reveal key interactions for drug design.

X-ray crystal structures at 2.9 Å resolution are reported for two complexes of catechol diethers with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles.