A Personal Perspective on Chemical Biology: Before the Beginning

Peter B. Dervan, Isr. J. Chem., 2019, 59, 71-83.

This perspective represents a brief personal account of early days before “chemical biology” emerged as a field of inquiry. Imagine a time when oligomers of DNA could not be synthesized and the order of the TACG letters in DNA could not be sequenced. Even the high resolution structure of the DNA double helix was not yet determined. 1975 was a time when there was a deep chasm between chemistry and biology. Chemists with precise knowledge of all the atoms in natural product architectures looked with dismay at the imprecise messy world of biology. Water was to be avoided! My view was that the power of synthetic organic chemistry should be used to create function, synthesis with a purpose. Our organic group at Caltech would embrace molecular recognition of biologics in water as a frontier for chemistry. We dreamed of inventing small molecules that would control the activity of macromolecules such as DNA, proteins and carbohydrates in living cells. We chemists would sky dive into the messy world of biology.


Molecular Recognition

Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands.

White S, Szewczyk JW, Turner JM, Baird EE, Dervan PB, 1998 Jan 29; 391(6666):468-71.

The design of synthetic ligands that read the information stored in the DNA double helix has been a long-standing goal at the interface of chemistry and biology. Cell-permeable small molecules that target predetermined DNA sequences offer a potential approach for the regulation of gene expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogues have been shown to interfere with gene expression, the triple-helix approach is limited to recognition of purines and suffers from poor cellular uptake. The subsequent development of pairing rules for minor-groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity. An Im/Py pair distinguishes G x C from C x G and both of these from A x T/T x A base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish AT from T x A. To break this degeneracy, we have added a new aromatic amino acid, 3-hydroxypyrrole (Hp), to the repertoire to test for pairings that discriminate A x T from T x A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of this third amino acid, hydroxypyrrole-imidazole-pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py and Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA.

see also: Trauger JW, Baird EE, Dervan PB.   Recognition of DNA by designed ligands at subnanomolar concentrations 1996 Aug 8; 382(6591):559-61.


A structural basis for recognition of A.T and T.A base pairs in the minor groove of B-DNA.

C L KielkopfS WhiteJ W SzewczykJ M TurnerE E BairdP B DervanD C Rees,   Science  1998; 282(5386):111-5.

(Kielkopf et al, 1998)Polyamide dimers containing three types of aromatic rings-pyrrole, imidazole, and hydroxypyrrole-afford a small-molecule recognition code that discriminates among all four Watson-Crick base pairs in the minor groove. The crystal structure of a specific polyamide dimer-DNA complex establishes the structural basis for distinguishing T.A from A.T base pairs. Specificity for the T.A base pair is achieved by means of distinct hydrogen bonds between pairs of substituted pyrroles on the ligand and the O2 of thymine and N3 of adenine. In addition, shape-selective recognition of an asymmetric cleft between the thymine-O2 and the adenine-C2 was observed. Although hitherto similarities among the base pairs in the minor groove have been emphasized, the structure illustrates differences that allow specific minor groove recognition.


Structural studies

Molecular recognition of the nucleosomal ‘‘supergroove’’

Edayathumangalam RS,   Weyermann P,   Gottesfeld JM,   Dervan PB,   Luger K.,  Proc Natl Acad Sci USA. 2004 May 4; 101(18):6864-9. Epub 2004 Apr 20.

Chromatin is the physiological substrate in all processes involving eukaryotic DNA. By organizing 147 base pairs of DNA into two tight superhelical coils, the nucleosome generates an architecture where DNA regions that are 80 base pairs apart on linear DNA are brought into close proximity, resulting in the formation of DNA ‘‘supergrooves.’’ Here, we report the design of a hairpin polyamide dimer that targets one such supergroove. The 2-Å crystal structure of the nucleosome–polyamide complex shows that the bivalent ‘‘clamp’’ effectively crosslinks the two gyres of the DNA superhelix, improves positioning of the DNA on the histone octamer, and stabilizes the nucleosome against dissociation. Our findings identify nucleosomal supergrooves as platforms for molecular recognition of condensed eukaryotic DNA. In vivo, supergrooves may foster synergistic protein–protein interactions by bringing two regulatory elements into juxtaposition. Because supergroove formation is independent of the translational position of the DNA on the histone octamer, accurate nucleosome positioning over regulatory elements is not required for supergroove participation in eukaryotic gene regulation.


Allosteric modulation of DNA by small molecules.

Chenoweth DM, Dervan PB, 2009 Aug 11; 106(32):13175-9


(Chenoweth and Dervan, 2009)Many human diseases are caused by dysregulated gene expression. The oversupply of transcription factors may be required for the growth and metastatic behavior of human cancers. Cell permeable small molecules that can be programmed to disrupt transcription factor-DNA interfaces could silence aberrant gene expression pathways. Pyrrole-imidazole polyamides are DNA minor-groove binding molecules that are programmable for a large repertoire of DNA motifs. A high resolution X-ray crystal structure of an 8-ring cyclic Py/Im polyamide bound to the central 6 bp of the sequence d(5'-CCAGGCCTGG-3')2 reveals a 4 A widening of the minor groove and compression of the major groove along with a >18 degrees bend in the helix axis toward the major groove. This allosteric perturbation of the DNA helix provides a molecular basis for disruption of transcription factor-DNA interfaces by small molecules, a minimum step in chemical control of gene networks.

see also: D. M. Chenoweth and P. B. Dervan, Structural Basis for Cyclic Py-Im Polyamide Allosteric Inhibition of Nuclear Receptor Binding J. Am. Chem. Soc.2010132 (41), pp 14521–14529


Molecular Engineering/Nanotechnology

Programming Multiple Protein Patterns on a Single DNA Nanostructure

Cohen JD,   Sadowski JP,   Dervan PB.   2008 Jan 16; 130(2):402-3.

The ability to create assemblies of proteins with spacing on the nanometer scale has important implications for proteomics, biodetection, and self-assembly. Structural DNA nanotechnology has led to the creation of a variety of nanostructures which should be capable of serving as an addressable template for the creation of complex molecular assemblies. The goal of such systems is to be able to position proteins or other components in distinct patterns with precise spacing. These systems take advantage of the well-defined structure and spacing of DNA and use these properties to act as a template for secondary components in a bottom-up approach toward self-assembly. Previous work in this area has primarily focused on the use of chemical or structural modifications of the DNA template in order to attach or recruit proteins or nanoparticles. We have recently shown that a single polyamide-biotin conjugate is capable of binding to a DX array made from two tiles without any modification of the target DNA. The highly programmable nature of pyrrole−imidazole polyamides make them particularly attractive for targeting specific DNA sequences. We demonstrated how the programmability of polyamide conjugates can be used to target orthogonal binding sites on a four tile DX-array. This allows us to use polyamides to arrange proteins into multiple distinct patterns using a common 2-D DNA template.

see also: Cohen JD, Sadowski JP, Dervan PB, Addressing single molecules on DNA nanostructures. 2007; 46(42):7956-9.


Regulation of gene expression

Suppression of androgen receptor-mediated gene expression by a sequence-specific DNA-binding polyamide

Nickols NG,   Dervan PB.   2007 Jun 19; 104(25):10418-23. Epub 2007 Jun 12.

Androgen receptor (AR) is essential for the growth and progression of prostate cancer in both hormone-sensitive and hormone-refractory disease. A DNA-binding polyamide that targets the consensus androgen response element binds the prostate-specific antigen (PSA) promoter androgen response element, inhibits androgeninduced expression of PSA and several other AR-regulated genes in cultured prostate cancer cells, and reduces AR occupancy at the PSA promoter and enhancer. Down-regulation of PSA by this polyamide was comparable to that produced by the synthetic antiandrogen bicalutamide (Casodex) at the same concentration. Genome-wide expression analysis reveals that a similar number of transcripts are affected by treatment with the polyamide and with bicalutamide. Direct inhibition of the AR-DNA interface by sequence-specific DNA binding small molecules could offer an alternative approach to antagonizing AR activity.

N. Nickols


In-vivo testing in mice

Antitumor activity of a pyrrole-imidazole polyamide.

Yang F,   Nickols NG,   Li BC,   Marinov GK,   Said JW,   Dervan PB.,   2013 Jan 29;110(5):1863-8. doi: 10.1073/pnas.1222035110. Epub 2013 Jan 14.

Many cancer therapeutics target DNA and exert cytotoxicity through the induction of DNA damage and inhibition of transcription. We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Polyamide treatment activates p53 signaling in LNCaP prostate cancer cells without detectable DNA damage. Genome-wide mapping of RNAP2 binding shows reduction of occupancy, preferentially at transcription start sites, but occupancy at enhancer sites is unchanged. Polyamide treatment results in a time- and dose-dependent depletion of the RNAP2 large subunit RPB1 that is preventable with proteasome inhibition. This polyamide demonstrates antitumor activity in a prostate tumor xenograft model with limited host toxicity.


Animal Toxicity of Hairpin Pyrrole-Imidazole Polyamides Varies with the Turn Unit.

Yang F,   Nickols NG,   Li BC,   Szablowski JO,   Hamilton SR,   Meier JL,   Wang CM,   Dervan PB,   2013 Sep 26; 56(18): 7449-7457. doi: 10.1021/jm401100s. Epub 2013 Sep 9.

A hairpin pyrrole-imidazole polyamide (1) targeted to the androgen receptor consensus half-site was found to exert antitumor effects against prostate cancer xenografts. A previous animal study showed that 1, which has a chiral amine at the α-position of the γ-aminobutyric acid turn (γ-turn), did not exhibit toxicity at doses less than 10 mg/kg. In the same study, a polyamide with an acetamide at the β-position of the γ-turn resulted in animal morbidity at 2.3 mg/kg. To identify structural motifs that cause animal toxicity, we synthesized polyamides 1−4 with variations at the α- and β-positions in the γ-turn. Weight loss, histopathology, and serum chemistry were analyzed in mice post-treatment. While serum concentration was similar for all four polyamides after injection, dose-limiting liver toxicity was only observed for three polyamides. Polyamide 3, with an α-acetamide, caused no significant evidence of rodent toxicity and retains activity against LNCaP xenografts.



Design of sequence-specific DNA-binding molecules.

Dervan PB,   1986 Apr 25; 232(4749):464-71.

Base sequence information can be stored in the local structure of right-handed double-helical DNA (B-DNA). The question arises as to whether a set of rules for the three-dimensional readout of the B-DNA helix can be developed. This would allow the design of synthetic molecules that bind DNA of any specific sequence and site size. There are four stages of development for each new synthetic sequence-specific DNA-binding molecule: design, synthesis, testing for sequence specificity, and reevaluation of the design. This approach has produced bis(distamycin)fumaramide, a synthetic, crescent-shaped oligopeptide that binds nine contiguous adenine-thymine base pairs in the minor groove of double-helical DNA.


Affinity cleavage method

see also: Taylor JS, Schultz PG, Dervan PB, DNA Affinity Cleaving. Sequence Specific Cleavage of DNA by Distamacyin-EDTA-Fe(II), Tetrahedron, 40, 457 (1984) see also: Schultz PG, Taylor JS, Dervan PB, Design and Synthesis of a Sequence Specific DNA Cleaving Molecule. (Distamycin-EDT)iron(II). J. Am. Chem. Soc. 104, 6861 (1982)


Footprinting methods for analysis of pyrrole-imidazole polyamide/DNA complexes.

Trauger JW,   Dervan PB 2001; 340:450-66.

Quantitative footprinting titration analyses.(left)Cleavage pattern generated by quantitative DNase I footprinting titration on a 3’-labeled DNA fragment in the presence of increasing ligand concentration (right). Langmuir binding titration isotherm obtained from DNase I data.

see also: Van Dyke MW, Dervan PB, Echinomycin Binding Sites on DNA, Science, 225, 1122 (1984)


Quantitative Microarray Profiling of DNA-Binding Molecules

Puckett JWMuzikar KATietjen J,   Warren CL,   Ansari AZ,   Dervan PB,   2007 Oct 10; 129(40):12310-9. Epub 2007 Sep 19.

A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5'-TACGT-3' within the hypoxia response element (HRE) yielded a CSI microarray-derived sequence motif of 5'-WWACGT-3' (W = A,T). A linear beta-linked polyamide programmed to target a (GAA)3 repeat yielded a CSI microarray-derived sequence motif of 5'-AARAARWWG-3' (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of Ka values across the microarray intensity spectrum.


Guiding the Design of Synthetic DNA-Binding Molecules with Massively Parallel Sequencing.

Meier JL,   Yu AS,   Korf I,   Segal DJ,   Dervan PB,   2012 Oct 24; 134(42):17814-22. doi: 10.1021/ja308888c. Epub 2012 Oct 10.

Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5'-WCGCGW-3' from 5'-WGCGCW-3'. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.


Sequence-specific cleavage of double helical DNA by triple helix formation.

Moser HE,   Dervan PB,   1987 Oct 30; 238(4827):645-50.

Homopyrimidine oligodeoxyribonucleotides with EDTA-Fe attached at a single position bind the corresponding homopyrimidine-homopurine tracts within large double-stranded DNA by triple helix formation and cleave at that site. Oligonucleotides with EDTA.Fe at the 5' end cause a sequence specific double strand break. The location and asymmetry of the cleavage pattern reveal that the homopyrimidine-EDTA probes bind in the major groove parallel to the homopurine strand of Watson-Crick double helical DNA. The sequence-specific recognition of double helical DNA by homopyrimidine probes is sensitive to single base mismatches. Homopyrimidine probes equipped with DNA cleaving moieties could be useful tools for mapping chromosome

see also: Strobel SA, Dervan PB, Site-Specific Cleavage of a Yeast Chromosome by Oligonucleotide-Directed Triple Helix Formation. Science, 249, 73 (1990)

see also: Beal PA, Dervan PB, Second Structural Motif for Recognition of DNA by Oligonucleotide-Directed Triple Helix Formation. Science, 251, 1360 (1991)