Anti-Mitotic Agents

More Vinca Alkaloids

1) All admin by IV = often as part of combination regimen 2) Share many common adverse effects a) Myelosuppression b) Peripheral neuropathy c) Hair loss d) Diarrhea e) Nausea 3) All are CYP3A4 substrates, PgP substrates a) Polymorphisms impact efficacy, tolerance b) Resistance develops by efflux overexpression There are a number of closely related vinca alkaloids Only 3 are approved in the US, although many other have seen clinical trials, and some are used routinely in other countries Vinblastine and vincristine are both natural products They differ only in the R group (Me vs CHO) Vinorelbine is a semisynthetic analog Note unsaturation in the upper (catharanthine) heterocycle Despite chemical similarity, there are differences in toxicity and antitumor efficacy Vincristine is used more commonly to treat children than adult cancers = pediatric cancers are more sensitive to it, and it is better tolerated at pediatric doses VRL is approved in the US for lung cancers = alone or in combination with cisplatin It is also active against breast and ovarian cancers As a class, the vinca alkaloids share common adverse effects = typically myelosuppression, peripheral neuropathy, diarrhea, nausea, etc They are all CYP3A4 substrates and they are good PgP substrates = resistance can evolve by transport upregulation (PgP overexpression) Student will NOT be held to recall these structural details of these (and other) antimitotics, but they should be able to recognize & identify the pharmacophores by structure

Eribulin

1) Fully synthetic analog of a natural product Halachondrin B found in marine sponges 2) Also binds to microtubules near the Vinca site and inhibits (+) = end MT growth 3) Admin IV to treat metastatic breast cancer 4) A liposomal formulation, and Mab-conjugate are under clinical study Approved in 2010 to treat patients with metastatic breast cancer who have received prior therapies (taxanes, anthracycline) that failed Approved in 2016 for inoperable liposarcoma (extends survival by 2x) The most common adverse reactions (in at least 25% of patients) associated with eribulin were neutropenia, anemia, asthenia/fatigue, alopecia, peripheral neuropathy, nausea, and constipation The most common serious adverse reactions reported in patients treated with eribulin were febrile neutropenia (4%) and neutropenia (2%) Peripheral neuropathy was the most common toxicity leading to discontinuation of eribulin (5%)

Summary

1) Microtubules dynamics are targeted by a number of important anti-cancer agents 2) Multiple drug classes delineated by characteristics of tubulin binding a) Inhibitors of MT growth = Vinca alkyloids b) Stabilizers of MT (inhibitors of MT Dynamics) = Taxanes, epothilones, estamustine 3) Know the important agents in each class

Vinca Alkaloids

1) Natural products used in folk medicine 2) Dimeric fusion of 2 heterocyclic cores Idole core (top) and dihydroindole (bottom) 3) Bind to microtubules = not tubulin 4) Bioactivity in diabetes, myelosuppression 5) VBL and VCR marketed in 1963 by Eli Lilly Having been used a lot as a folk remedy for centuries, studies in the 1950s showed that C. roseus contained 70 alkaloids, many of which are biologically active While initial studies for its use in diabetes were disappointing, the discovery that it caused myelosuppression (decreased activity of the bone marrow) led to its study in mice with leukemia, whose lifespan was prolonged by the use of vinca preparation Vinblastin was isolated in 1958 Approved by FDA in 1963 These molecules bind more tightly to microtubule-associated tubulin, than to isolated tubulin Vincrstine was approved by the FDA in July 1963 as Oncovin marketed by Lilly There are a number of closely related Vinca Alkyloids

Paclitaxel Semisynthesis

1) Paclitaxel and analogs can be prepared semi-synthetically from 10-decetylbaccatin III isolated from yew needles 2) Taus baccata now grown in farms A solution came with the realization that paclitaxel and other analogs can be prepared by semisynthesis beginning with 10-deacetylbaccatin III (shown here) It is actually abundant in the needles = not just the bark---of the European Yew Taus Baccata This yew is now grown in tree farms & harvested for the isolation of the paclitaxel precursor

Paclitaxel Supply

1) Paxlitaxel extracted from the bark of the Pacific Yew 2) Sufficient for clinical trials, but mass production by this method threatened whole forests 3) Total synthesis not practical When clinical trials of paclitaxel were about to wrap up in 1992, there was no clear path forward for the production of the drug that seemed viable Enough drug for trials had been extracted from the bark of the Pacific Yew, but stripping bark from mature trees killed them, and the thought of killing whole forests for drug production was not welcomed by environmentalists The total synthesis of the drug was also out of the question It had not been achieved in 1992; let alone scaled for production

Objectives

1) Understand the role that microtubles (MT) and MT dynamics plays in mitosis 2) Learn of anti-mitotic agents used in cancer therapy that interfere with these dynamics

Taxane Properties Problematica

1) Very poor bioavailability 2) Not soluble in water 3) Nephrotoxicity 4) Neutropenia 5) Does not cross BBB 6) Drug resistance can emerge quickly via transporter up-regulation

Centrosome-mediated microtubule nucleation

As cells transition from interphase to mitosis, distinct events occur to prepare for chromosome segregation = chromosomes condense, nuclear envelop breakdown (NEBD) occurs, the duplicated centrosomes separate and move to opposite sides of the cell and the spindle assembles (a) As cells transition from G2 to mitosis, the microtubule-nucleating capacity of the centrosomes is increased, which is coupled with increased microtubule dynamics Each centrosome nucleates mirotubule arrays, which radiate dynamic microtubule plus ends toward the cell center where they contact the chromosomes Microtubules search the cellular space as they grow & shrink, covering several micrometers and varying their trajectories during each growth period owing to their inherent dynamic instability

Chromatin-mediated microtubule nucleation

Chromatin generates a localized biochemical environment that supports the nucleation & growth of microtubules that become incorporated into the spindle through different mechanisms A number of proteins have now been identified that are important participants in microtubules nucleation, stabilization, and crosslinking Chromatin-mediated microtubule nucleation pathways increase the density of microtubules around kinetochores, which facilitates their capture & association with the centrosome-initiated microtubules It is not important to know the details, but RAN (a Ras-like protein) facilitates nucleation & recruits Aurora B to the kinetechore

Colchicine Analogs Under Investigation

Colchicine = NP, not used for cancer (gout), important for early understanding of tubulin function in 1970s ZD6126 is a mimic of colchicine and it is Phase I clinical trial = also found to have antivascular effects well below mtd ZD6126 is a novel vascular-targeting agent that disrupts the endothelial tubulin cytoskeleton causing selective occlusoin of tumor vasculature and extensive tumor necrosis This Phase I clinical study was conducted to evaluate the dose and admin schedule of ZD6126 Combretstatin is a NP isolated from a willow tree bark from South Africa and clinical trails turned out to be too toxic CA4P is a prodrug of CA4 has been studied extensively, but is still not approved for use (withdrawn from Phase III) Oxigene (CA4P) received orphan drug status in 2016 for gioma (and 7 years marketing exclusivity upon approval) The rest are modified forms based on combretstatin Tularik compounds irreversibly bind tubulin; alkylate tubulin Cys 239, not a substrate for P-glycoprotein Batabulin in Phase II T900607 in Phase I

Colchicine Binding Site

Colchicine is not used in oncology therapy, but has been used to learn much about microtubule dynamics The colchicine binding site is beta-tubulin at the alpha, beta-tubulin dimer interface, near the non-exchangeable nucleotide binding site It binds to alpha, beta-tubulin dimers, which are then incorporated into microtubles with inhibited growth potential Microtubules assembled from alpha, beta-tubulin with colchicine bound adopt a more curved shape, and cannot adopt a straight structure because of steric clashes introduced at the alpha/beta interface Colchicine thereby prevents normal tubulin growth and assembly into K-fibers

The Mitotic Checkpoint

Crucial regulators of the mitotic checkpoint at the kinetochore are shown On each kinetochore, when microtubules are captured and tension in these is detected, the checkpoint is satisfied and MAD2 is released, providing the biochemical signal to proceed to anaphase CDC = cell division cycle CENPE = centromere protein E MAD = mitotic arrest deficient

Estramustine

Estramustine accumulates in prostate tissues Estramustine is a conjugate of an alkylating agent (non-nitrogen mustard) and a 17-beta-estradiol It is actually formulated as the phosphate prodrug that is rapidly dephosphorylated in the intestinal tract to expose estramustine It was originally synthesized and evaluate as a DNA alkylating agent that would accumulate in estrogen-receptor bearing breast cancer cells, but proved inactive in clinical trials It was later shown that it does NOT alkylate DNA It does accumulate in the prostate, however, by binding to a protein named teh estramustine-binding protein (EMBP), and was shown to be active clinically Several mechanisms of estramustine activity have been studied, but the principle mechanism of action seems to be as an inhibitor of microtubule dynamics It binds to beta-tubulin, but not at the vinca or colchicine sites---and only to some tubulin isoforms, and seems to have an effect similar to the taxanes and epolilones Estramustine phosphate is used for the treatment of progressive prostate cancers

Gemcitabine/Abraxane Approved as First-Line Combination for Patients with Metastatic Pancreatic Cancer 2013

First new treatment option in 8 years Pancreatic cancers are some of the most difficult to treat This graph summarizes results from the MPACT study, which compared 2 patient cohorts = one receiving Gemcitibine (the pyrimidine nucleotide RR inhibitor) alone, to another receiving Gemcitibine and albumin-paclitaxel The long-term prognosis for either group is not good, but for a majority of patients---survival is extended by 2 to 3 months

Effect of Tubulin Binding Drugs

For drug molecules that bind to different sites, they induce different changes to the mitotic spindle Shown on this slide is the mitotic spindle of the cells treated with the tubulin binding agents and viewed by microscopy Colchichine completely destroys the mitotic spindle, the vinblastine also leads to mitotic spindle change Paclitaxel actually stabilizes the spindle Epothilone also stabilizes the mitotic spindle

Next Generation Taxanes

Formulations to improve solubility, distribution, and resistance 1) Liposomal paclitaxel formulations allow an increase in the maximum tolerated dose a) Lipusu commercialized b) EndoTAG-1 and LEP-ET in trials 2) Paclitaxel PEG conjugates XYOTAX = studed, but not approved 3) Paxlitaxel-eluting stents (TAXUS = used in cardiovascular surgery 4) ABRAXANE = albumin bound paclitaxel a) Maximum tolerated dose of paclitaxel is higher when delivered to bound albumin b) Response rate in clinical trials is approx twice as high for patients given paclitaxel alone c) Approved by FDA in 2005 for drug-resistant breast cancer d) Indications since expanded to non-small cell lung cancer in 2012

Epothilones

Ixabepilone, Azaepothilone B, Ixempra = Breast cancers Peripheral neuropathy and neutropenia are common side effects Epothilones are another class of microtubule stabilizers that belong to the macrolide drug family Initially isolated from bacterial sources for their unique antifungal & cytotoxic activities Epothilines appear to compete for binding at the paclitaxel binding site in the interior of assembled microtubules A number of epothilones have been studied in different clinical applications from Alzheimer's (epothilone D) to cancer Active compounds vary the epoxide (B), and the heterocycle (A) and its substituents Little tolerance for changes in the macrocycle One is approved for anti-cancer therapy; Ixabepilone (azaepothilone B) Approved in 2007 for use in metastatic breast cancer that are resistant to anthracyclines and/or taxanes Typically used in combination with capecitabine Generation of epothilone resistant cancer cell lines in lab is much more difficult than for taxol

Colchicine

Long used to treat gout, arrests microtubule assembly and inhibits many cellular functions At nanomolar concentrations, colchicine blocks neutrophil adhesion dependent on microtubule assembly, and inhibits monosodium urate crystal-induced inflammation Cycotchrome P450 3A4, the multidrug transporter P-glycoprotein, and the drugs that bind these proteins influence its pharmacokinetics and pharmacodynamics Trial evidence supports its efficacy in acute gout and in preventing gout flares, but it has a narrow therapeutic index, and overdosage is associated with GI, hepatic, renal, neuromuscular, and cerebral toxicity, bone marrow damage, and high mortality

Mitosis and Microtubules

Microtubule structures (stained green) undergo marked morphological changes to mediate specific functions throughout the cell cycle Microtubule dynamics vary during the cell cycle, being least dynamic in interphase cells and most dynamic during mitosis, making this an important target for anticancer drug design Most tubulin-binding agents (TBAs) act on spindle microtubule dynamics, which are important for normal spindle function Disruption of spindle microtubules results in mitotic arrest, which can lead to cell death through various mechanism DNA is shown in blue

Microtubule Assembly

Microtubules are hollow tubes assembled from heterodimers or alpha-tubulin and beta-tubulin in GTP-dependent manner Microtubules are hollow tubes of about 250 angstroms in diameter that are assembled from alpha-tubulin-beta-tubulin (alpha-beta tubulin) heterodimers in a GTP-dependent manner The tubulin subunits make 2 types of filament contacts: Longitudinal contacts run the length of the microtubule forming protofilaments, and lateral contacts between protofilaments (generally alpha-tubulin to alpha-tubulin and beta-tubulin to beta-tubulin) form the circumference of the microtubule Longitudinal contacts are GTP-mediated Non-exchangeable GTP occupies the alpha-subunit = promotes dimerization with beta GTP/GDP exchange on beta subunit and mediates linear (longitudinal growth) The MT is polar = with (+) to which new tubulin and be added, and a (-) end from with tubulin can be removed Microtubule growth and disassembly is a dynamic process It occurs slowly spontaneously, but extension can occur rapidly with assembly onto a preformed nucleus A nucleus for MT growth in mitosis is on the centrosomes Growth and disassembly is also facilitated (and controlled) by other Microtubule-Associated Proteins (MAPs) The idea that microtubules self-assemble in cells is highly oversimplified

Taxanes, more

Paclitaxel first approved in 1992 On the WHO list of 100 essential medicines Used to treat a variety of metastatic tumors Docetaxel approved for use in 1995 Docetaxel is slightly more water soluble than paclitaxel, and appear to have 2x better binding affinity to microtubules, although additional potency has little therapeutic benefit Cabazitaxel approved for prostate cancers---particularly those which have resisted treatment with docetaxel It is NOT as efficiently transported by P-glycoprotein, available by IV only Typically given with prednisone Severe neutropenia limits use As a class, principal toxicity is neutropenia

Taxanes

Taxanes bind in the interior of microtubules and stabilize them to disassembly Taxanes have unique mechanism of action, and broad antitumor activity that makes them one of the more important classes of anti-cancer agents The first interest in taxanes emerged in 1963 when extracts from the bark of the Pacific Yew tree were shown to have impressive activity in tumor models In 1971, paclitxel (in picture) was identified as the active agent Paclitaxel (Taxol) and its analogs are now known to bind to beta-tubulin in the interior of assembled microtubules where it stabilizes their quaternary structure and inhibits microtubule disassembly Recall, microtubule strand dynamics (growth/disassembly/regrowth) is critical to the random search for chromosomes Paclitaxel analogs do not prevent the binding of other antimitotics to microtubules Resistance arises with increased production of P-glycoprotein

The Mitotic Spindle

The mitotic spindle has a crucial role in ensuring the accurate segregation of chromosomes into the 2 daughter cells during cell division, which is paramount for maintaining genome integrity It is a self-organized & dynamic macromolecular structure that is constructed from microtubules, microtubule-associated proteins and motor proteins The spindle is a self-organized bipolar array of microtubules The minus ends of the microtubules are focused into the spindle poles, which is where the centrosomes reside to nucleate and organize spindle microtubules The microtubule plus ends radiate towards the equator, where microtubules from each pole overlab, resulting in an antiparallel array The chromosomes attach to the microtubules via kinetochores, which are multiprotein complexes that assemble on the centromere of each sister chromatid Once 20 to 30 kinetochore microtubules (K-MTs) attach to a kinetochore, they become stabilized into bundles called K-fibers *The dynamic growth, disassembly and regrowth of microtubules is essential to the successful construction of the spindle*

The Phases of Mitosis

The progression of mitosis through morphological stages is shown Upon entry into Prophase, the nuclear membrane dissolves and we see the emergence of centrosomes that form and become the focal point for the separation of chromosomes into 2 distinct sets Specific druggable protein targets that function during mitosis are highlighted Kinesin spindle protein (KSP) is required to establish mitotic spindle bipolarity by driving centrosome separation Centromeric protein E (CENPE) is required for accurate chromosome congression at metaphase during mitosis Kinase Aurora A is crucial for centrosome maturation & separation during early prophase Aurora B is a member of the chromosomal passenger complex (CPC) and is involved in histone H3 phosphorylation, chromosomal condensation, chromosomal alignment on the metaphase plate, bipolar centromere-microtubule attachments, spindle checkpoint, and cyokinesis During mitosis, Polo-like kinase-1 is involved in centrosome maturation and formation of the mitotic spindle Polo-like kinase 1 is also required for exit from mitosis and the separation of sister chromatids during anaphase Polo-like kinase 1 might also have a role in cytokinesis through the phosphorylation of the kinesin-like motor protein MKLP1 Each of these have been subjects of drug discovery efforts for many years, and many specific inhibitors are currently in clinical trials But, the common thread (pun intended) through all stages of mitosis is microtubules = shown in this figure as these little fibers---that provide the dominant morphological changes that accompany the progression of mitosis While there is hope ways can be found to interfere with microtubule control in the future, the important therapeutic agents of the present alter microtubule dynamics by directly biding to (or in) microtubules (Tublin binding agents; TBA) Drugs in development: Selective PLK1 inhibitors = volasertib, rigosertib, GSK461364 APC/C inhibitors = TAME Aurora A inhibitors = Alisertib; MK50108 Aurora B inhibitors = Barasertib Pan Aurora inhibitors = Danusertib

Mechanisms of Resistance to Tubulin Binding Agents

There are a number of ways that cancer cells can develop resistance to tubulin binding agents Information about resistance mechanisms can help inform the choice of alternative therapy options that remain effective These mechanisms can be broadly divided into 5 categories: a) Decreased cellular drug accumulation owing to the overexpression of membrane bound drug efflux proteins such as P-glycoprotein b) A direct alteration in the drug target through mutation, as is evident in drug resistant cell lines c) Altered expression of tubulin isotypes or microtubule-associated protein (MAP) expression that reduces drug efficacy, as is evident in drug-resistant cell lines & clinical samples d) Changes to the microtubules induced by interactions with or regulation by other cytoskeletal proteins, such as gamma-actin or actin-regulating proteins, that can affect the ability of tubulin binding agents to induce mitotic arrest and cell death e) Defects in apoptotic pathways also influence resistance to tubulin binding agents There are multiple alpha and beta tubulin isotypes that are encoded by different genes, located on different chromosomes and that have tissue and cell-specific expression patterns Pro and anti-apoptotic proteins, which include p53 and Bcl2 family proteins, are tethered or transported by microtubules Abnormal and high levels of beta-III tubulin, an isotype that is normally expressed at high levels in neuronal cells & testicular sertoli cells, has been associated with more aggressive and drug resistant cancers Patupilone was shown to be active in beta-III tubulin-expressing ovarian tumor cell lines, and molecular modelling of target binding suggests that it binds beta-III tubulin more effectively than paciltaxel

Antimitotic Peptides

There are also some extraordinarily potent peptide inhibitors of microtubule (postiive) end growth that are natural products The 2 compounds shown here = Maytansine & Dolastatin 10 are exmaples Maytansine is one representative of a class of macrocylic lactams (Maytansinoids) isolated from plants Dolastatins are produced by microbes in aquatic sea slugs = are some of the most potent antimitotics known Structural studies have revealed that these compounds also bind overlapping the Vinca binding site (although, because they are lager, their binding site is larger) Maytansine, Dolastatin 10 are too potent for clinical use (no therapeutic window), but they are good candidates for use in antibody-drug conjugates or other strategies that target cancer cells selectively Brentuximab and trastuzumab are examples of currently marketed antibody-drug conjugates Idea is that you use the antibody to selectively target only cancer cells = give the conjugates at much lower therapeutic doses where they can be tolerated

Vinca Alkaloid Binding Site

This is a view of beta-tubulin from the top (positive) end This is the vinca alkaloid binding site and the yellow one is the exchangeable nucleotide

Tubulin Binding Sites

To date, 3 drug binding sites have been identified: Vinca alkaloid site, colchicine site, and taxol site Vinca (green) overlaps exchangeable nucleotide site on beta-tubulin Colchicine (pink) overlaps with non-exchangeable nucleotide site at alpha-beta tubulin dimer interface Taxol (orange) = distinct site on beta-tubulin

Inhibitors of Microtubule Dynamics

Two mechanisms 1) Tubulin polymerization inhibitors a) Vinca alkaloids b) Antimitotic peptides c) Colchicine 2) Microtubule stabilizers a) Taxanes b) Epothilones There are 2 classes of tubulin-binding anticancer agents Polymerization inhibitors = block addition of dimers to growing positive end & continue to dissociate from negative end Depolymerization inhibitors = block the dissociation and promote and stabilize the formation of microtubules

Effect on microtubule dynamics

a) A few microtubules of vinblastine bound to high-affinity sites at the microtubule plus end suffice to suppress microtubule dynamics Suppresses positive end growth b) Colchicine forms complexes with tubulin dimers and copolymerizes into microtubule lattice, suppressing microtubule dynamics Binding results in a subtle (10 degree) shift in the alpha-beta orientation that alters the assembled microtubulue architecture c) A microtubule cut away to show the interior surface is shown Paclitaxel binds along the interior surface of the microtubule, suppressing its dynamics Actually stabilizes lateral assembly, slowing (or preventing) disassembly

Antimitotic agents stall

progression of cell cycle through various phases of Mitosis