Synthesis and structure-activity relationships of a novel series of HIV-1 protease inhibitors encompassing ABT-378 (Lopinavir)

The HIV protease inhibitor ABT-378 (Lopinavir) has a 2,6-dimethylphenoxyacetyl group in the P-2' position. Analogues in which this group is replaced with various substituted phenyl or heteroaryl groups were synthesized and the structure-activity relationships explored.     

Hits, Fhits and Nits: beyond enzymatic function

We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein-substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions.     

Novel lopinavir analogues incorporating heterocyclic replacements of six-member cyclic urea--synthesis and structure-activity relationships

The HIV protease inhibitor ABT-378 (lopinavir) has a six-member cyclic urea in the P-2 position. A series of analogues in which the six-member cyclic urea is replaced by various heterocycles was synthesized and the structure-activity relationships explored.     

SPR‐based plastic optical fibre biosensor for the detection of C‐reactive protein in serum

A plastic optical fibre biosensor based on surface plasmon resonance for the detection of C‐reactive protein (CRP) in serum is proposed. The biosensor was integrated into a home‐made thermo‐stabilized microfluidic system that allows avoiding any thermal and/or mechanical fluctuation and maintaining the best stable conditions during the measurements. A working range of 0.006–70 mg L^–1 and a limit of detection of 0.009 mg L^–1 were achieved. These results are among the best compared to other SPR‐based biosensors for CRP detection, especially considering that they were achieved in a real and complex medium, i.e. serum. In addition, since the sensor performances satisfy those requested in physiologically‐relevant clinical applications, the whole biosensing platform could well address high sensitive, easy to realize, real‐time, label‐free, portable and low cost diagnosis of CRP for future lab‐on‐a‐chip applications.

3D sketch (left) of the thermo‐stabilized home‐made flow cell developed to house the SPR‐based plastic optical fibre biosensor. Exemplary response curve (shift of the SPR wavelength versus time) of the proposed biosensor (right) for the detection of C‐reactive protein in serum.     

Non-Invasive Prenatal Chromosomal Aneuploidy Testing - Clinical Experience: 100,000 Clinical Samples

Objective

As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT.

Study Design

The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA–licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting.

Results

NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result.

Conclusion

NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.     

Influenza neuraminidase inhibitors: structure-based design of a novel inhibitor series

Combinatorial and structure-based medicinal chemistry strategies were used together to advance a lead compound with an activity of K(i) = 58 microM via a potency enhancement of >70 000-fold to an analogue with an activity of K(i) = 0.8 nM against influenza neuraminidase (A/Tokyo/67). Lead optimization was initiated using molecular modeling and combinatorial chemistry. Protein crystal structures revealed that inconsistent structure-activity relationship (SAR) data resulted from different binding orientations of the inhibitor core five-membered rings from one series to another. Binding modes for a series of compounds showed up to a 180 degrees variation in orientation of the five-membered ring within the active site. Potent analogues were only achieved with chemical series that were observed to bind in the same orientation and yielded consistent SAR. In one series, consistent binding was obtained by an unprecedented occupation of a negatively charged binding pocket by a neutral methyl ester unit. The structural rationale for this novel SAR variation, based on protein crystallographic data, is given.     

Synthesis and SAR studies of potent HIV protease inhibitors containing novel dimethylphenoxyl acetates as P2 ligands

Isopropyl substituted 4-thioazolyl valine side chains are highly optimized P(2)-P(3) ligands for C2 symmetry-based HIV protease inhibitors, as exemplified by the drug ritonavir. Replacement of the side chain with the conformationally constrained hexahydrofurofuranyloxy P(2) ligand in combination with a dimethylphenoxyacetate on the other end of the ritonavir core diamine yielded highly potent HIV protease inhibitors. The in vitro antiviral activity in MT4 cells increased by 10- and 20-fold, respectively, in the absence and presence of 50% human serum compared to ritonavir. The structure-activity relationships of inhibitor series with this combination of ligands were investigated. Preliminary pharmacokinetic studies in rats indicated rapid elimination of the inhibitors from the blood, and the plasma levels were not significantly enhanced by coadministration with ritonavir. However, the novel structural features and the high intrinsic antiviral potency of this series provides potential for the future exploration of prodrug strategies.     

Hepatitis C NS3 protease inhibition by peptidyl-alpha-ketoamide inhibitors: kinetic mechanism and structure

A series of novel peptidyl-alpha-ketoamide compounds were evaluated as inhibitors of the deltaNS3-NS4A serine protease from the hepatitis C virus. These peptidyl-alpha-ketoamide inhibitors with Ki values ranging from 0.17 nM to 5.6 microM exhibited slow-binding inhibition. Kinetic studies established one-step kinetic mechanisms and dissociation rate constants in the 3-7 x 10(-5) s(-1) range for these compounds. The association rate constants, which ranged from 10 to 263,000 M(-1) s(-1), were responsible for the greater than four order of magnitude overall binding affinity range exhibited by this series. An X-ray crystal structure of a protease-inhibitor complex revealed an unusual interaction between the oxyanion of the adduct and the protein as well as a significant movement in the S1' region of the protein loop comprising residues 35-42. These results are quite different from peptidyl-alpha-ketoacid inhibition of HCV protease, which reportedly undergoes no notable conformational changes and proceeds with a two-step slow-binding kinetic mechanism.