About

Derek Kinchington BSc. PhD, FRCPath

Dr. Kinchington was awarded a Fellowship of the Royal College of Pathologists in 2014 on the basis of Published Works.

In 1984 the etiological agent of AIDS was discovered to be a retrovirus. This began the drive to discover compounds which would inhibit the replication of the Human Immunology Virus (HIV). Dr. Derek Kinchington joined Professor Donald Jeffries at the Virology Department of St Mary’s Hospital Medical School in 1986 to head a small group to develop the techniques necessary to discover novel compounds active against HIV. Derek worked with Roche Products UK Ltd and with other medicinal chemistry groups, mostly within the UK university sector, sponsored by the then newly established MRC AIDS Directed Programme.

 Techniques for evaluating potential antiviral drugs.

T- Cell lines from oncology laboratories were available for testing of novel compounds. These transformed cell lines were easier to maintain but the separation and maintenance of primary cells was more involved and required extensive procedures to isolate them in high purity from peripheral blood or lymphoid tissue ^{2, 5}. As the structure and function of the HIV genome was unravelled, the range of anti-HIV targets grew. These included compounds which acted on the surface of T-cells to block virus entry, nucleosides and nucleotides which inhibited the early event of viral DNA formation and later events such as the integration of viral DNA into chromosomal DNA and the maturation of viral particles³⁹. By adding compounds at specific times post-infection it was possible to infer whether a compound acted early or late in the replication cycle. For example, nucleosides and protease inhibitors added immediately post-infection showed good activity against HIV replication. In contrast, at 24 hours post-infection nucleosides lost most of their activity but the protease inhibitors were still potent inhibitors of HIV ^{2, 4, 9}_.

Protease inhibitors.

In 1987 Roche Products started their programme of synthesising transition-state competitive inhibitors targeted at the large polyproteins gag p55 and gag-pol p160. The rationale was that these inhibitors would bind to the active site of the HIV proteinase enzyme. They would compete with the natural amino acid sequences of HIV which span the cleavage sites within gag-pol substrate; thus cleavage into the smaller proteins would not occur and mature virions would not form. Those immature virions, which lacked core structures, would not infect other circulating T-cells. This maturation process is complex and is initiated by a random, concentration controlled auto-catalytic event whereby two large gag polyproteins associate, are cleaved and form a HIV – protease molecule. Once a single proteinase molecule has formed, a cascade occurs producing many proteinase molecules.

The first assays used to measure antiviral activity were based on syncytia produced when infected and uninfected cells were mixed. Counting replicate wells with more than two or three compounds at a time proved impractical. Further, it did not give the sensitivity that would be needed to investigate a large number of compounds. A number of new assays which tested for HIV p24 in blood had recently come onto the market and these were compared for sensitivity and dose response ¹¹. It was found that the Coulter assay performed best for our use. A low infectious dose of virus could be used and the incubation period was accurately controlled. This enabled ten or more compounds and controls to be tested simultaneously. This report was the first published in comparing the use of these assays for drug development¹¹.

In October 1989, after approximately 250 compounds were synthesised, the compound Ro 31-8959 (XVII) ^{3, 10} was selected as the most potent. In laboratory cells lines the IC50  was less than 0.4 nM, the IC50 was greater than 10,000 nM and the selectivity was therefore greater than 25,000. The data was published in April 1990 in Science. Dr Noel Roberts and Dr Joseph Martin at Roche products Ltd were responsible for the Biology and Medicinal Chemistry respectively, and Dr Kinchington carried out the Virology studies at  Mary’s Hospital Medical School.

Compound XVII (saquinavir) was licensed in 1995.

XVII was found to be active against HIV-1, HIV-2, AZT- resistant strains of HIV-1, SIV and against T-cells chronically infected with HIV-1. The latter result is important as nucleosides show little activity in chronically infected cells ⁹. Further, virus particles centrifuged from supernatants from cell cultures infected with HIV-1 and treated with XVII, when resuspended in fresh culture fluid regained their infectivity because the compound diffused out from the virions. This observation supported the concept and highlighted the need to maintain drug concentration in patients at physiological levels. Saquinavir, although clinically very effective, had poor bioavailability and was administered to patients in high concentration. It needed the next generation of protease inhibitors for this problem to be overcome.

In 1993 Dr. Kinchington worked with a group from St. Andrew’s which had synthesised a number of transition-state protease inhibitors that had good cell uptake properties. Some of these compounds were active against HIV-1 but generally had low activity and low selectivity⁸_.

Nucleotide inhibitors.

Roche Products had purchased the rights from NIH in 1986 to develop ddC and Roche chemists also synthesised a series of mono- and di-fluoro analogues ^{12, 13}. These were the first compounds Derek worked with having joined St. Mary’s Hospital Medical School. Activity of these nucleoside analogues was compared using syncytia assays. Zalcitabine (ddC) was used in the clinic alone or in combination with zidovudine before the new proteinase inhibitors were introduced but is infrequently prescribed now.

Nucleoside inhibitors.

In 1988 through the MRC AIDS Directed Programme Derek collaborated with Professor Christopher McGuigan, then at Southampton University Chemistry Department. He had started work on synthesising a novel series of nucleoside inhibitors of reverse transcriptase, targeted to HIV. The fundamental concept was to eliminate the first kinase dependent activation step necessary to convert inactive nucleosides into the final active tri-phosphates. The second and third kinase steps to produce the di-phosphate and the tri-phosphate molecules are most often not rate limiting. This concept had two advantages: firstly cell membranes with low or zero kinase activity could be penetrated by these compounds by a kinase by-pass mechanism. Secondly it might be possible to deliver much less of the compound to the cell and get the same anti-HIV activity. One problem with this approach is that cell membranes are impermeable to nucleoside monophosphates so novel masked phosphate compounds were synthesised so as allow the molecule to enter cells freely. Thus a large number of AZT analogues were evaluated using the various assays that had been developed at St. Mary’s ^{14 -30}_.

This was a very productive relationship: the data and ways to optimise the experimental conditions were discussed on a regular basis. This work did not result in a drug against HIV at the time, but latterly Professor McGuigan’s work on kinase by-pass did produce a very active compound against HCV, that reached phase II clinical trials, and a related agent for the treatment of pancreatic cancer. This promising early data was reported at the 2013 meeting of the American Society of Clinical Oncology.

In 1990 there were three laboratories which were part of the MRC effort to evaluate novel compounds active against HIV. The laboratories used a range of assays, virus strains and cell lines. In order to compare the results between the centres a number of standard compounds were tested by them. A laboratory at the Rega Institute, Leuven was invited to participate. Overall there were broad similarities in the results from the different centres. However, some key discrepancies were found which reinforced the need for a range of assays ³².

Virucidal agents.

Early in the HIV epidemic virucidal agents were considered as an alternative to antiviral therapy in poorer countries. In 1992 at St. Bartholomew’s Medical College a study was carried out to test such agents in low pH and in the presence of genital secretions_. A novel assay was devised to investigate their effects ³¹. Interestingly in this system seminal plasma was most effective in reducing HIV infectivity.

Cell membranes as targets for chemotherapy.

During this period Dr. Kinchington also worked with several other groups investigating novel targets for chemotherapy. Data showed that cells infected with HIV resulted in changes in the fatty acid composition of cell membranes ^{36, 37}. In a related study the polyunsaturated fatty acid, lithium g-linolenate (Li-GLA) was shown to kill selectively HIV-infected cells probably by enhanced lipid peroxidation of the cell membranes ³⁵. In another study, comparing herpesviruses, HIV and a picornavirus it was proposed that the effect of 2-hydroxymyristic acid on the inhibition of myristoylation, a’ virus-essential’ cellular pathway, may be another route for blocking virus replication³⁴.

Antisense oligodeoxynucleotides.

A study was carried out in collaboration with groups from Copenhagen and Georgetown University, USA to investigate the property of antisense oligodeoxynucleotides as inhibitors of HIV-1. Assays were carried using both acutely – and chronically -infected cells. The activity of these compounds is primarily related to strand length and not specificity. Blocking of cell receptors used by HIV to gain entry into cells is the most likely mode of action of these compounds ³³.

Immunomodulators.

Starting in 1993 Derek worked with a small company who were investigating the use of immunomodulators in the treatment of HIV infections and AIDS. He was responsible for setting up the biological assays needed. Initially fifteen compounds were investigated in an anti-CD3-stimuulated T lymphoblastic cell proliferation assay. One compound the sodium salt of 2 chloro-5-nitrobenzoic acid (Na –CNBA) was chosen as the lead compound and caused CD4+ cell proliferation probably via the IL-2 pathway³⁸. Further studies were carried out on isolated CD4+ cells from healthy human donors and from HIV patients. In collaboration with the United States Food and Drug Administration he planned a study using SCIDhu-HIV infected mice. A significant increase in CD4+ cells was observed in mice treated with Na-CNBA plus antiviral drugs compared with those just treated with antiviral drugs alone. Two large scale clinical studies were initiated in 1995 to investigate the effect of IL-2 on patients with CD4+ cells of 50 -299 cells /ul (SILCAAT) and another where CD4+ cells were greater than 300/ul ( ESPIRIT ). These trials ran from 1999 -2009 in 25 countries, and although CD4+ cell numbers increased in patients there was no improvement in the numbers of memory cells. The data for Na-CNBA from donor CD4+ cells and in the SCIDhu model, to date, has not been published. There is still a question as to whether an increase in memory cells would have been achieved with Na- CNBA.

Poxvirus studies.

Dr. Kinchington’s first project in virology was working with Professor Keith Dumbell in comparing the genomes of smallpox and human monkeypox. He carried out some of the restriction enzyme mapping of the cloned sequences. He also developed the techniques for heteroduplex analysis and the electronic measurement of DNA molecules photographed obtained by electron microscopy. This work highlighted key genome sequences, which were absent in human monkeypox, when compared to those in smallpox virus ^{6, 7}. These smallpox sequences were later shown to be responsible for its virulence.

Cancer Studies
At the Imperial Cancer Research Fund (now Cancer UK) Dr Kinchington worked on two aspects of cancer. Firstly, with Professor Beverley Griffin, on EBV and its association with Burkitt’s lymphoma he developed the methodology for spreading the EBV DNA, and a system to measure concatamers observed in the images obtained by electron microscopy ⁴². Later studies showed that these defective EBV circles are involved in the induction of viral early antigens and in virion formation.

In Professor Michael Waterfield’s group Derek helped to map the EGF receptor gene by using heteroduplex mapping. The gene has coding sequences of 5.5kb (introns) located in total gene sequence of 110 kb (introns plus exons). His contribution was to identify the position of the intron/exon boundaries along the length of the gene. Heteroduplex analysis of the small cDNA clone (5.5kb) annealed with a number of larger genomic clones (15- 25 kb)   located these boundaries to within twenty base pairs. He subsequently carried out some of the DNA -sequencing of the appropriate regions within the genomic clones to identify the specific sequences at the exon-intron boundaries ^{40, 41}. The epidermal growth factor (EGF) receptor, when altered, can serve as a transforming protein and members of the EGF receptor family are over expressed in a high percentage of human breast cancers.

In 1997 Derek worked with a company interested in a number of novel furanones as anticancer agents. He set up several cell culture assays which ran in parallel and discriminated between the differential cytotoxic effects of the compounds under test ^{43, 44, 45}.