Ralph Laufer

Discovery of Raltegravir, a Potent, Selective Orally Bioavailable HIV-Integrase Inhibitor for the Treatment of HIV-AIDS Infection

Human immunodeficiency virus type-1 (HIV-1) integrase is one of the three virally encoded enzymes required for replication and therefore a rational target for chemotherapeutic intervention in the treatment of HIV-1 infection. We report here the discovery of Raltegravir, the first HIV-integrase inhibitor approved by FDA for the treatment of HIV infection. It derives from the evolution of 5,6-dihydroxypyrimidine-4-carboxamides and N-methyl-4-hydroxypyrimidinone-carboxamides, which exhibited potent inhibition of the HIV-integrase catalyzed strand transfer process. Structural modifications on these molecules were made in order to maximize potency as HIV-integrase inhibitors against the wild type virus, a selection of mutants, and optimize the selectivity, pharmacokinetic, and metabolic profiles in preclinical species. The good profile of Raltegravir has enabled its progression toward the end of phase III clinical trials for the treatment of HIV-1 infection and culminated with the FDA approval as the first HIV-integrase inhibitor for the treatment of HIV-1 infection.

Highly selective agonists for substance P receptor subtypes

The existence of a third tachykinin receptor (SP‐N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N‐methylation of individual peptide bonds in the C‐terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl‐[Asp6,Me‐Phe8]SP6‐11, elicits half‐maximal contraction of the guinea pig ileum through the neuronal SP‐N receptor at a concentration of 0.5 nM. At least 60,000‐fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP‐P and SP‐E). The action of selective SP‐N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP‐N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.

Enhancing B- and T-Cell Immune Response to a Hepatitis C Virus E2 DNA Vaccine by Intramuscular Electrical Gene Transfer

ABSTRACT We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8+ T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Behavioural effects of receptor-specific substance P agonists

&NA; Septide and senktide are synthetic substance P (SP) agonists with extremely high selectivity for 1 of the 3 known SP receptor subtypes. When injected intrathecally, they produced dramatically different behavioural effects. Septide, the selective SP‐P receptor agonist, evoked intense, compulsive scratching, biting and licking of the hind limb, with no sign of motor flaccidity, and without measurable effect on responses to noxious thermal or mechanical stimulation of the foot or tail. In contrast, senktide, the selective SP‐N receptor agonist, produced profound, but transient, motor flaccidity, reduced response to noxious stimuli and, at low doses, ‘wet‐dog shakes.’ These various symptoms, all previously associated with SP and/or synthetic SP analogues, appear therefore to derive from activation of distinct SP receptor subtypes.

Potent Inhibitors of Subgenomic Hepatitis C Virus RNA Replication through Optimization of Indole-N-Acetamide Allosteric Inhibitors of the Viral NS5B Polymerase

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC(50) = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.

CNTF VARIANTS WITH INCREASED BIOLOGICAL POTENCY AND RECEPTOR SELECTIVITY DEFINE A FUNCTIONAL SITE OF RECEPTOR INTERACTION

Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand‐specific alpha‐receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (LIFR). At high concentrations, CNTF can also activate the LIFR and possibly other cross‐reactive cytokine receptors in the absence of CNTFR alpha. To gain a better understanding of its structure‐function relationships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display technology. Variants with greatly increased CNTFR alpha affinity were selected from a phage‐displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix. Selected variants contained substitutions of the wild‐type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evidence for an important functional role of the mutagenized region in CNTFR alpha binding. Affinity enhancing mutations conferred to CNTF increased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contrast, the same mutations did not potentiate the CNTFR alpha‐independent receptor actions of CNTF. These CNTF analogs thus represent receptor‐specific superagonists, which should help to elucidate the mechanisms underlying the pleiotropic actions of the neurocytokine.

Discovery of (7R)-14-Cyclohexyl-7-{(2-(dimethylamino)ethyl)(methyl) amino}-7,8-dihydro-6H- indolo(1,2-e)(1,5)benzoxazocine-11-carboxylic Acid (MK-3281), a Potent and Orally Bioavailable Finger-Loop Inhibitor of the Hepatitis C Virus NS5B Polymerase †

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

Design and synthesis of bicyclic pyrimidinones as potent and orally bioavailable HIV-1 integrase inhibitors.

HIV integrase is one of the three enzymes encoded by HIV genome and is essential for viral replication, but integrase inhibitors as marketed drugs have just very recently started to emerge. In this study, we show the evolution from the N-methylpyrimidinone structure to bicyclic pyrimidinones. Introduction of a suitably substituted amino moiety modulated the physical-chemical properties of the molecules and conferred nanomolar activity in the inhibition of spread of HIV-1 infection in cell culture. An extensive SAR study led to sulfamide (R)- 22b, which inhibited the strand transfer with an IC50 of 7 nM and HIV infection in MT4 cells with a CIC95 of 44 nM, and ketoamide (S)- 28c that inhibited strand transfer with an IC50 of 12 nM and the HIV infection in MT4 cells with a CIC95 of 13 nM and exhibited a good pharmacokinetic profile when dosed orally to preclinical species.

Identification of novel, selective, and stable inhibitors of class II histone deacetylases. Validation studies of the inhibition of the enzymatic activity of HDAC4 by small molecules as a novel approach for cancer therapy.

5-Aryl-2-(trifluoroacetyl)thiophenes were identified as a new series of class II HDAC inhibitors (HDACi). Further development of this new series led to compounds such as 6h, a potent inhibitor of HDAC4 and HDAC6 (HDAC4 WT IC(50) = 310 nM, HDAC6 IC(50) = 70 nM) that displays 40-fold selectivity over HDAC1 and improved stability in HCT116 cancer cells (t(1/2) = 11 h). Compounds 6h and 2 show inhibition of alpha-tubulin deacetylation in HCT116 cells at 1 microM concentration and antiproliferation effects only at concentrations where inhibition of histone H3 deacetylation is observed.

Direct determination of unbound intrinsic drug clearance in the microsomal stability assay

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. Determination of the unbound intrinsic clearance (CLin,u) is essential for the accurate comparison of compounds, since nonspecific binding to microsomes can lead to an underestimation of the microsomal clearance. In this study, a new method (linear extrapolation in the stability assay, LESA) was established, which allows direct calculation of CLin,u from microsomal stability data, without the need to independently determine the fraction of free (unbound) drug. The method was validated using nine drugs with different chemical structures and physicochemical properties. The CLin,u of these compounds was extrapolated from the intrinsic clearance values obtained at different concentrations of human liver microsomes and compared with that calculated by the conventional method, using microsomal intrinsic clearance values and the free fraction of drug determined by equilibrium dialysis, ultracentrifugation, or ultrafiltration. A good agreement was observed between the data generated by the LESA method and those determined by conventional procedures. The method was further evaluated using a published dataset for 10 additional drugs and found to yield intrinsic clearance data comparable to the previously reported values. LESA provides a convenient and rapid method to determine the influence of microsome binding on intrinsic clearance in a single assay.