Patrick T. Flaherty

Design and Optimization of Renin Inhibitors: Orally Bioavailable Alkyl Amines

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC(50) of 0.47nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.

Optimization of orally bioavailable alkyl amine renin inhibitors.

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension.

Identification of HIV Inhibitors Guided by Free Energy Perturbation Calculations

Free energy perturbation (FEP) theory coupled to molecular dynamics (MD) or Monte Carlo (MC) statistical mechanics offers a theoretically precise method for determining the free energy differences of related biological inhibitors. Traditionally requiring extensive computational resources and expertise, it is only recently that its impact is being felt in drug discovery. A review of computer-aided anti-HIV efforts employing FEP calculations is provided here that describes early and recent successes in the design of human immunodeficiency virus type 1 (HIV-1) protease and non-nucleoside reverse transcriptase inhibitors. In addition, our ongoing work developing and optimizing leads for small molecule inhibitors of cyclophilin A (CypA) is highlighted as an update on the current capabilities of the field. CypA has been shown to aid HIV-1 replication by catalyzing the cis/trans isomerization of a conserved Gly-Pro motif in the Nterminal domain of HIV-1 capsid (CA) protein. In the absence of a functional CypA, e.g., by the addition of an inhibitor such as cyclosporine A (CsA), HIV-1 has reduced infectivity. Our simulations of acylurea-based and 1-indanylketone-based CypA inhibitors have determined that their nanomolar and micromolar binding affinities, respectively, are tied to their ability to stabilize Arg55 and Asn102. A structurally novel 1-(2,6-dichlorobenzamido) indole core was proposed to maximize these interactions. FEP-guided optimization, experimental synthesis, and biological testing of lead compounds for toxicity and inhibition of wild-type HIV-1 and CA mutants have demonstrated a dose-dependent inhibition of HIV-1 infection in two cell lines. While the inhibition is modest compared to CsA, the results are encouraging.

Design, synthesis, and testing of an 6-O-linked series of benzimidazole based inhibitors of CDK5/p25

Alzheimers disease (AD) is a progressive neurodegenerative disease resulting in cognitive and behavioral impairment. The two classic pathological hallmarks of AD include extraneuronal deposition of amyloid β (Aβ) and intraneuronal formation of neurofibrillary tangles (NFTs). NFTs contain hyperphosphorylated tau. Tau is the major microtubule-associated protein in neurons and stabilizes microtubules (MTs). Cyclin dependent kinase 5 (CDK5), when activated by the regulatory binding protein p25, phosphorylates tau at a number of proline-directed serine/threonine residues, resulting in formation of phosphorylated tau as paired helical filaments (PHFs) then in subsequent deposition of PHFs as NFTs. Beginning with the structure of Roscovitine, a moderately selective CDK5 inhibitor, we sought to conduct structural modifications to increase inhibitory potency of CDK5 and increase selectivity over a similar enzyme, cyclin dependent kinase 2 (CDK2). The design, synthesis, and testing of a series of 1-isopropyl-4-aminobenzyl-6-ether-linked benzimidazoles is presented.

Cyclin-Dependent Kinase 5 (CDK5) Controls Melanoma Cell Motility, Invasiveness, and Metastatic Spread—Identification of a Promising Novel therapeutic target

Despite considerable progress in recent years, the overall prognosis of metastatic malignant melanoma remains poor, and curative therapeutic options are lacking. Therefore, better understanding of molecular mechanisms underlying melanoma progression and metastasis, as well as identification of novel therapeutic targets that allow inhibition of metastatic spread, are urgently required. The current study provides evidence for aberrant cyclin-dependent kinase 5 (CDK5) activation in primary and metastatic melanoma lesions by overexpression of its activator protein CDK5R1/p35. Moreover, using melanoma in vitro model systems, shRNA-mediated inducible knockdown of CDK5 was found to cause marked inhibition of cell motility, invasiveness, and anchorage-independent growth, while at the same time net cell growth was not affected. In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases. Inhibition of lung metastasis was further validated in a syngenic murine melanoma model. CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype. Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells. Thus, experimental data presented here provide strong evidence for a crucial role of aberrantly activated CDK5 in melanoma progression and metastasis and establish CDK5 as promising target for therapeutic intervention.

Biphenyl/diphenyl ether renin inhibitors: Filling the S1 pocket of renin via the S3 pocket

Structure-based design led to the discovery of a novel class of renin inhibitors in which an unprecedented phenyl ring filling the S1 site is attached to the phenyl ring filling the S3 pocket. Optimization for several parameters including potency in the presence of human plasma, selectivity against CYP3A4 inhibition and improved rat oral bioavailability led to the identification of 8d which demonstrated antihypertensive efficacy in a transgenic rat model of human hypertension.

Identification of benzimidazole-based inhibitors of the mitogen activated kinase-5 signaling pathway

The MEK-signaling pathways are complex but critical signaling cascades that correlate an extracellular signaling event with internal cell processes. To date at least seven MEK isozymes have been identified. MEK5, in particular, is upregulated in multiple forms of tumors. Analysis of the EGF-induced MEK5 signaling cascade in cultured HEK cells has identified compounds that can inhibit MEK5 phosphorylation of ERK5; observed biological activity is dependent on chemical variation.

Oncogenic signaling of MEK5-ERK5

Mitogen-activated protein kinases (MAPKs) regulate diverse cellular processes including proliferation, cell survival, differentiation, and apoptosis. While conventional MAPK constituents have well-defined roles in oncogenesis, the MEK5 pathway has only recently emerged in cancer research. In this review, we consider the MEK5 signaling cascade, focusing specifically on its involvement in drug resistance and regulation of aggressive cancer phenotypes. Moreover, we explore the role of MEK5/ERK5 in tumorigenesis and metastatic progression, discussing the discrepancies in preclinical studies and assessing its viability as a therapeutic target for anti-cancer agents.

Structure–activity relationships of benzimidazole-based selective inhibitors of the mitogen activated kinase-5 signaling pathway

In a prior communication we identified a novel class of benzimidazole-based inhibitors of EGF-induced phosphorylation of ERK5. In this paper we examine the biological activity of several 1-isopropyl-4-amino-6-ether linked benzimidazole-based compounds for their ability to selectively inhibit EGF-mediated ERK5 phosphorylation; potential utility of variation at the 6-position was indicated by the initial structural feature survey. Modification of EGF-induced formation of pERK1/2 and pERK5 in HEK293 cells were analyzed by Western blot analysis. Subsequent analysis of selected compounds in a high-throughput multiple kinase scan and the NCI 60-cell-line screen is presented.

N-Acetyl-l-Cysteine Protects Astrocytes against Proteotoxicity without Recourse to Glutathione

N-acetyl-l-cysteine (NAC) exhibits protective properties in brain injury models and has undergone a number of clinical trials. Most studies of NAC have focused on neurons. However, neuroprotection may be complemented by the protection of astrocytes because healthier astrocytes can better support the viability of neurons. Here, we show that NAC can protect astrocytes against protein misfolding stress (proteotoxicity), the hallmark of neurodegenerative disorders. Although NAC is thought to be a glutathione precursor, NAC protected primary astrocytes from the toxicity of the proteasome inhibitor MG132 without eliciting any increase in glutathione. Furthermore, glutathione depletion failed to attenuate the protective effects of NAC. MG132 elicited a robust increase in the folding chaperone heat shock protein 70 (Hsp70), and NAC mitigated this effect. Nevertheless, three independent inhibitors of Hsp70 function ablated the protective effects of NAC, suggesting that NAC may help preserve Hsp70 chaperone activity and improve protein quality control without need for Hsp70 induction. Consistent with this view, NAC abolished an increase in ubiquitinated proteins in MG132-treated astrocytes. However, NAC did not affect the loss of proteasome activity in response to MG132, demonstrating that it boosted protein homeostasis and cell viability without directly interfering with the efficacy of this proteasome inhibitor. The thiol-containing molecules l-cysteine and d-cysteine both mimicked the protective effects of NAC, whereas the thiol-lacking molecule N-acetyl-S-methyl-l-cysteine failed to exert protection or blunt the rise in ubiquitinated proteins. Collectively, these findings suggest that the thiol group in NAC is required for its effects on glial viability and protein quality control.